Otospora bareai, a new fungal species in the Glomeromycetes from a dolomitic shrub land in Sierra de Baza National Park (Granada, Spain)

A new fungal species of the Glomeromycetes was isolated from the rhizosphere of Pterocephalus spathulatus and Thymus granatensis, two rare endemic plants growing on dolomite in the Sierra de Baza (Granada, southern Spain). The fungus was propagated in pot cultures of Sorghum vulgare and Trifolium pratense for 4 y and it is described here on the basis of the spores found in nature and formed in pot cultures. Its brown spores (140-210 microm diam) form laterally on a persistent, brown stalk (=neck) of a sporiferous saccule. They have two walls without ornamentation: a brown, three- to four-layered outer wall and a hyaline two- to three-layered inner wall. The unique combination of spore formation and spore wall structure does not fit with any of the known fungal genera. Spore formation is similar to that of Acaulospora spp. and Archaeospora trappei, but Acaulospora spp. has three spore walls with a characteristic "beaded" wall, and the outer wall of Ar. trappei is simple, thin, hyaline and only bilayered. Spore wall structure of the new fungus is similar to that of Entrophospora infrequens, however this fungus forms its spores internally, inside the hyphal stalk of the sporiferous saccule. Molecular analyses of the small subunit of the ribosomal gene phylogenetically place the new fungus next to Diversispora spurca, which forms one-walled glomoid spores (i.e. terminally on hyphae). Based on these analyses we place the new fungus into a new genus in the family Diversisporaceae under the epithet Otospora bareai.     

Ambispora granatensis, a new arbuscular mycorrhizal fungus, associated with Asparagus officinalis in Andalucia (Spain)

A new dimorphic fungal species in the arbuscular mycorrhiza-forming Glomeromycota, Ambispora granatensis, was isolated from an agricultural site in the province of Granada (Andalucía, Spain) growing in the rhizosphere of Asparagus officinalis. It was propagated in pot cultures with Trifolium pratense and Sorghum vulgare. The fungus also colonized Ri T-DNA transformed Daucus carota roots but did not form spores in these root organ cultures. The spores of the acaulosporoid morph are 90-150 μm diam and hyaline to white to pale yellow. They have three walls and a papillae-like rough irregular surface on the outer surface of the outer wall. The irregular surface might become difficult to detect within a few hours in lactic acid-based mountings but are clearly visible in water. The structural central wall layer of the outer wall is only 0.8-1.5 μm thick. The glomoid spores are formed singly or in small, loose spore clusters of 2-10 spores. They are hyaline to pale yellow, (25)40-70 μm diam and have a bilayered spore wall without ornamentation. Nearly full length sequences of the 18S and the ITS regions of the ribosomal gene place the new fungus in a separate clade next to Ambispora fennica and Ambispora gerdemannii. The acaulosporoid spores of the new fungus can be distinguished easily from all other spores in genus Ambispora by the conspicuous thin outer wall.     

Taxonomic revision transferring species in Kuklospora to Acaulospora (Glomeromycota) and a description of Acaulospora colliculosa sp. nov. from field collected spores

In a phylogenetic study of arbuscular mycorrhizal fungal species in Acaulospora (Acaulosporaceae, Glomeromycota) we discovered that species classified in genus Kuklospora, a supposed sister clade of Acaulospora, did not partition as a monophyletic clade. Species in these two genera can be distinguished only by the position of the spore relative to a precursor structure, the sporiferous saccule, as either within (entrophosporoid) or laterally (acaulosporoid) on the saccule subtending hypha. Subsequent spore differentiation follows identical patterns and organization. Molecular phylogeny reconstructed from nrLSU gene sequences, together with developmental data, support the hypothesis that the entrophosporoid mode of spore formation evolved many times and thus represents a convergent trait of little phylogenetic significance. Therefore genus Kuklospora is rejected as a valid monophyletic group and it is integrated taxonomically into genus Acaulospora. Thus Acaulospora colombiana and Acaulospora kentinensis are erected as new combinations (formerly Kuklospora colombiana and Kuklospora kentinensis). Mode of spore formation is demoted from a genus-specific character to one that is included with other traits to define Acaulospora species. In addition we describe a new AM fungal species, Acaulospora colliculosa (Acaulosporaceae), that originated from a tallgrass prairie in North America. Field-collected spores of A. colliculosa are small (<100 μm diam), hyaline or subhyaline to pale yellow and form via entrophosporoid development based on structure and organization of cicatrices and attached hyphae. Each spore consists of a bilayered spore wall and two bilayered inner walls. A germination orb likely forms after the completion of spore development to initiate germination, but this structure was not observed. A character distinguishing A. colliculosa from other Acaulospora species is hyaline to subhyaline hemispherical protuberances on the surface of the outer spore wall layer. A phylogeny reconstructed from partial nrLSU gene sequences unambiguously placed A. colliculosa in the Acaulospora clade.     

Glomus africanum and G. iranicum, two new species of arbuscular mycorrhizal fungi (Glomeromycota)

Two new arbuscular mycorrhizal fungal species (Glomeromycota) of genus Glomus, G. africanum and G. iranicum, are described and illustrated. Both species formed spores in loose clusters and singly in soil and G. iranicum sometimes inside roots. G. africanum spores are pale yellow to brownish yellow, globose to subglobose, (60-)87(-125) μm diam, sometimes ovoid to irregular, 80-110 x 90-140 μm. The spore wall consists of a semipermanent, hyaline, outer layer and a laminate, smooth, pale yellow to brownish yellow, inner layer, which always is markedly thinner than the outer layer. G. iranicum spores are hyaline to pastel yellow, globose to subglobose, (13-)40(-56) μm diam, rarely egg-shaped, prolate to irregular, 39-54 x 48-65 μm. The spore wall consists of three smooth layers: one mucilaginous, short-lived, hyaline, outermost; one permanent, semirigid, hyaline, middle; and one laminate, hyaline to pastel yellow, innermost. Only the outermost spore wall layer of G. iranicum stains red in Melzer's reagent. In the field G. africanum was associated with roots of five plant species and an unrecognized shrub colonizing maritime sand dunes of two countries in Europe and two in Africa, and G. iranicum was associated with Triticum aestivum cultivated in southwestern Iran. In one-species cultures with Plantago lanceolata as the host plant G. africanum and G. iranicum formed arbuscular mycorrhizae. Phylogenetic analyses of partial SSU sequences of nrDNA placed the two new species in Glomus group A. Both species were distinctly separated from sequences of described Glomus species.     

Six new species of microsporidia of the genus Amblyospora (Microspora: Amblyosporidae) from blood sucking mosquitoes (Diptera: Culicidae) from the west Siberia

Microsporidia of the genus Amblyospora parasiting the adipose body of mosquito larvae of the genus Aedes and Culex has been studied with both light and electron microscopy. Six new species of microsporidia are described based on ultrastructural characteristics of spores and sporogony stages. Amblyospora flavescens sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 165 nm. Exospore is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, large vesicular, lamellar. The anisofilar polar filament with 10--11 coils (3 1/2 + 2 1/2 + 4-5). Fixed spores are 6.3 +/- 0.1 x 4.24 +/- 0.1 microm. Amblyospora kolarovi sp. n. Mature spores are egg-shaped. The spore wall with three layers, about 265-315 nm. Exospore shapes tucks on the surface of spore. It is two-membranous. Subexospore is quagge, structural. Endospore is electron-translucent. Polaroplast consists of two parts: lamellar and large vesicular. The anisofilar polar filament with 11-13 coils (3 + 8-10). Fixed spores are 5.4-5.6 x 3.5-4.2 microm. Amblyospora orbiculata sp. n. Mature spores are widely egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 155 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is absent. Endospore is electron-translucent. Polaroplast consists of three parts: lamellar, vesicular, lamellar. Polar filament is anisofilar, with 11 1/2 coils (4 1/2 + 1 + 6). Fixed spores are 6.3 +/- 0.1 x x 4.0 +/- 0.1 microm. Amblyospora rugosa sp. n. Mature spores are egg-shaped. On a back pole there is a small concavity. The spore wall with three layers, about 225 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 17 1/2 coils (3 1/2 + 1 + 13). Fixed spores are 5.3 +/- 0.1 x 3.7 +/- 0.1 microm. Amblyospora undata sp. n. Mature spores are egg-shaped. The spore wall is three-layered, about 220 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. The anisofilar polar filament with 8 coils (3 + 5). Fixed spores are 5.0 +/- 0.1 x 3.0 +/- 0.1 microm. Amblyospora urski sp. n. Mature spores have widely oval form. The back pole is concave. The spore wall with three layers, about 280 nm. Exospore is shapes tucks on a surface of spore. It is two-membranous. Subexospore is quaggy, structural. Endospore is electron-translucent. Polaroplast lamellate. Polar filament is anisofilar, with 6 coils (2 + 4). Fixed spores are 4.4 +/- 0.1 x 2.9 +/- 0.1 microm.     

Development of Acaulospora rehmii spore and hyphal swellings under root-organ culture

A strain of Acaulospora rehmii was, for the first time, successfully grown in vitro on Ri T-DNA transformed carrot roots allowing the in situ observation of Acaulospora spore development and of extraradical thin-walled hyphal swellings. The sporogenous hypha developed intercalarly along thin-walled coenocytic hyphae. The distal part of the sporogenous hypha swelled slightly as a sporiferous saccule primordium followed by the differentiation of a lateral spore primordium along the neck of the sporogenous hypha. Both structures matured simultaneously, and the sporiferous saccule began to collapse after spore maturation and complete differentiation of the spore wall. Several of the in situ observations on in vitro differentiated A. rehmii spores are concordant with previous ontogenic studies done on other Acaulospora species obtained from in vivo cultures. New and original observations on the early developmental stages of sporiferous saccules and spores and on the occurrence of small diameter intercalary hyphal swellings provide additional elements in the study of Acaulospora sporulation process and life cycle.     

ULTRASTRUCTURAL STUDIES OF IN SITU DEVONIAN SPORES: BARINOPHYTON CITRULLIFORME

Each sporangium in the Upper Devonian taxon Barinophyton citrulliforme contains both microspores and megaspores. Microspores range up to 50 μm in diam and possess a homogeneous sporoderm characterized by an outer separable layer. The sporoderm of the megaspores (up to 900 μm) is constructed of sporopollenin units that are loosely arranged in the outer portion of the wall, and that give the megaspore wall a spongy organization. Ultrastructural evidence suggests that the small spores were not abortive megaspores, but that both spore types were functional. The spores of this plant, as well as other Devonian spores that show less dramatic size differences, are suggested as demonstrating a phase in the evolution of heterospory where sex determination was established in spores within the same sporangium prior to the evolution of micro- and megasporangia.     

A new species of Myxozoa, Henneguya rondoni n. sp. (Myxozoa), from the peripheral nervous system of the Amazonian fish, Gymnorhamphichthys rondoni (Teleostei)

Henneguya rondoni n. sp. found in the peripheral lateral nerves located below the two lateral lines of the fish Gymnorhamphichthys rondoni (Teleostei, Rhamphichthyidae) from the Amazon river is described using light and electron microscopy. Spherical to ellipsoid cysts measuring up to 110 microm in length contained only immature and mature spores located in close contact with the myelin sheaths of the nervous fibres. Ellipsoidal spores measured 17.7 (16.9-18.1)-microm long, 3.6 (3.0-3.9)-microm wide, and 2.5 (2.2-2.8)-microm (n=25) thick. The spore body measuring 7.0 (6.8-7.3)-microm long was formed by two equal symmetric valves, each with an equal tapering tail 10.7 (10.3-11.0) microm in length. The tails were composed of an internal dense material surrounded by an external homogeneous sheath of hyaline substance. The valves surrounded two equal pyriform polar capsules measuring 2.5 (2.2-2.8)-microm long and 0.85 (0.79-0.88)-microm (n=25) wide and a binucleated sporoplasm cell containing globular sporoplasmosomes 0.38 (0.33-0.42) microm (n=25) in diam. with an internal eccentric dense structure with half-crescent section. Each polar capsule contains an anisofilar polar filament with 6-7 turns obliquely to the long axis. The matrix of the polar capsule was dense and the wall filled with a hyaline substance. The spores differed from those of previously described species. Based on the ultrastructural morphology of the spore and specificity to the host species, we propose a new species name H. rondoni n. sp.     

Differential cycles of range contraction and expansion in European high mountain plants during the Late Quaternary: insights from Pritzelago alpina (L.) O. Kuntze (Brassicaceae)

Nuclear DNA sequence variation of the internal transcribed spacer (ITS) and amplified fragment length polymorphisms (AFLPs) were used to illuminate the evolutionary history of Pritzelago alpina, a herbaceous perennial of (sub)alpine to nival habitats of the European high mountains. Maximum likelihood analysis of ITS sequences of P. alpina, Hornungia petraea and Hymenolobus procumbens (the 'Pritzelago alliance') resolved P. alpina and H. petraea as sister taxa. ITS divergence estimates support an origin for P. alpina in the Late Tertiary, while intraspecific diversification started in the Late Quaternary (0.4-0.9 million years ago). AFLP analysis of 76 individuals of P. alpina, representing 24 localities across its entire west-east distribution, identified four mountain lineages in Cantabria, the Pyrenees, (south-) western Alps, and northeastern Alps/Tatras/Carpathians. In an analysis of molecular variance (amova), 14.3% of the total variation derived from this separation. However, relationships among these lineages remained unresolved in neighbour-joining and principal co-ordinates analyses, suggesting a population history of near simultaneous vicariance. Comparison with our previous ITS/AFLP study of Anthyllis montana (Fabaceae) indicates that the two co-distributed but altitudinally differentiated plant species exhibit temporally concordant but spatially discordant patterns of genetic variation. Moreover, levels of AFLP divergence were significantly lower in P. alpina than in the submediterranean, lower-elevation A. montana. Together, these data are consistent with a 'displacement refugia model', which predicts that European mountain plant species associated with lower- and upper-elevation habitats had a different cycle of range contraction into (long-term) glacial and (short-term) interglacial refugia, respectively.     

Spore ornamentation of Haplosporidium pickfordi Barrow, 1961 (Haplosporidia), a parasite of freshwater snails in Michigan, USA

Spore ornamentation is increasingly recognized as a key character for species differentiation and genus assignment in the phylum Haplosporidia. Unfortunately, spore ornamentation is known for only a small number of described species so it is difficult to assign most species to genera with any confidence. Scanning and transmission electron microscopy were used to determine the presence and morphology of spore ornamentation of Haplosporidium pickfordi collected from the digestive gland of the snail Physella parkeri in Douglas Lake, Michigan. Spores possess filaments that are derived from the spore wall and originate from two separate areas at the posterior end of the spore. When spores are first isolated from host tissue, filaments are fused into a sheet that wraps around the spore, passing under the opercular lid. These filaments gradually unravel when spores are held in water and after about 14 d most filaments project freely from the posterior end of the spore. The number of filaments could not be determined with certainty, but appears to be approximately nine. Filaments are 100 nm in diam. and up to 50 microm in length. The presence of spore wall-derived filaments confirms the placement of the parasite in the genus Haplosporidium.     

A new species of Haplosporidium Caullery & Mesnil, 1899 in the marine false limpet Siphonaria lessonii (Gastropoda: Siphonariidae) from Patagonia

A new species of Haplosporidium Caullery & Mesnil, 1899 parasitising the pulmonate gastropod Siphonaria lessonii Blainville in Patagonia, Argentina, is described based on morphological (scanning and transmission electron microscopy) and sequence (small subunit ribosomal RNA gene) data. Different stages of sporulation were observed as infections disseminated in the digestive gland. Haplosporidium patagon n. sp. is characterised by oval or slightly subquadrate spores with an operculum that is ornamented with numerous short digitiform projections of regular height, perpendicular to and covering its outer surface. The operculum diameter is slightly larger than the apical diameter of the spore. Neither the immature nor mature spores showed any kind of projections of the exosporoplasm or of the spore wall. Regarding phylogenetic affinities, the new species was recovered as sister to an undescribed species of Haplosporidium Caullery & Mesnil, 1899 from the polychaete family Syllidae Grube from Japanese waters. The morphological characters (ornamentation of the operculum, spore wall structure, shape and size of spores, and the lack of spore wall projections) corroborate it as an as yet undescribed species of Haplosporidium and the first for the phylum in marine gastropods of South America. Siphonaria lessonii is the only known host to date.     

Fine structure of the myxosporean,Henneguya curimata n. sp., parasite of the Amazonian fish,Curimata inormata (Teleostei,Curimatidae)

Henneguya curimata n. sp. (Myxozoa, Myxobolidae) is described from the kidney of the teleost Curimata inormata collected in an estuarine region of the Amazon River, near Belém. Brazil. This myxosporean produces large cysts (0.6-1.2 mm in diam.) that represent plasmodia containing all life cycle stages, including spores. The spore body is ellipsoidal (approximately 16.6 microm in length and approximately 6.2 microm in width), and each valve presents a tapering tail (approximately 19.1 microm in length). These valves surround the binucleate sporoplasm cell and two ellipsoidal polar capsules located side-by-side at the same level, measuring 6.5 x 1.2 microm each and containing 10-11 coils of the polar filament. On the basis of its host specificity and on data collected by light and electron microscopy, the organism, H. curimata n. sp. is distinguished as a new species. The taxonomic affinities and morphological comparisons with other similar species of the same genus are discussed.     

BASIDIOSPOROGENESIS IN BOLETUS RUBINELLUS. I. STERIGMAL INITIATION AND EARLY SPORE DEVELOPMENT

Sterigmal initiation in Boletus rubinellus resembled hyphal tip growth. Four stages in early basidiospore development have been delineated based on gross morphology, and changes in wall layers and cytoplasm. Changes in wall layers and cytoplasm during spore development were stage-specific. During Stage 1 the spore wall consisted of two layers identical to those of the sterigmal wall with occasional pellicle remnants on the outer surface. The onset of wall differentiation began in Stage 2, and during Stage 3 wall layers characteristic of the mature spore developed. At Stage 4 there was a pronounced gradient in wall thickness from the apex to the base of the spore. Small vesicles (30–60 nm diam) were uniformly distributed in the cytoplasm of spherically enlarging spores (Stage 2), but during spore elongation (Stages 3 and 4) numerous larger vesicles as well as small vesicles aggregated at the spore apex. A variety of cytoplasmic organelles entered the spore during Stage 3; however, migration of storage materials and the nucleus to the spore did not occur until late basidiospore development. The hilar appendix body developed in the earliest spore primordium and persisted until Stage 3. Development of wall layers and their differential thickening, distribution of vesicles, and probable function of the hilar appendix body are discussed with reference to the control of spore shape. Systematic implications of the data are considered.     

Myxobolus insignis sp. n. (Myxozoa, Myxosporea, Myxobolidae), a parasite of the Amazonian teleost fish Semaprochilodus insignis (Osteichthyes, Prochilodontidae)

A new myxosporean species is described from the fish Semaprochilodus insignis captured from the Amazon River, near Manaus. Myxobolus insignis sp. n. was located in the gills of the host forming plasmodia inside the secondary gill lamellae. The spores had a thick wall (1.5-2 microm) all around their body, and the valves were symmetrical and smooth. The spores were a little longer than wide, with rounded extremities, in frontal view, and oval in lateral view. They were 14.5 (14-15) microm long by 11.3 (11-12) microm wide and 7.8 (7-8) microm thick. Some spores showed the presence of a triangular thickening of the internal face of the wall near the posterior end of the polar capsules. This thickening could occur in one of the sides of the spore or in both sides. The polar capsules were large and equal in size surpassing the midlength of the spore. They were oval with the posterior extremity rounded, and converging anteriorly with tapered ends. They were 7.6 (7-8) microm long by 4.2 (3-5) microm wide, and the polar filament formed 6 coils slightly obliquely to the axis of the polar capsule. An intercapsular appendix was present. There was no mucous envelope or distinct iodinophilous vacuole.     

Coltriciella minuscula sp. nov., a new species of poroid fungus on Pinus merkusii from an Indonesian tropical forest

Coltriciella minuscula sp. nov. is described and illustrated from a specimen collected on Pinus merkusii from Bogor, Indonesia. This species is characterized by the size of its basidiocarp up to 4 mm in diam, with long hair on the stipe and with ornamented spores. Both morphological distinctiveness and phylogenetic separation based upon analyses of nrDNA ITS sequences support the establishment of this new species. Morphological dissimilarities between C. minuscula and closely related species are discussed.     

Ultrastructural Changes During Germination of Dictyostelium discoideum Spores

Spores of Dictyostelium discoideum undergo significant changes in fine structure during germination. The mitochondria progressively become less dense and lose their peripherally attached ribosomes, and the tubuli become more pronounced as germination proceeds. During this period, the three-layered spore wall breaks down in two stages: first, the outer and middle layers are ruptured as a unit, and, second, the inner wall is breached. Crystals and dark (lipid) bodies disappear shortly before or during emergence of the myxamoebae. Autophagic vacuoles are found in dormant spores and throughout the entire germination process. The addition of cycloheximide to germinating spores inhibited the loss of the crystals and dark (lipid) bodies. In addition, the drug inhibited the breakdown of the inner wall layer. Cycloheximide did not prevent the formation of the water expulsion vesicle or the apparent function of the autophagic vacuoles.     

Observations on the life stages of Sphaerothecum destruens n. g., n. sp., a mesomycetozoean fish pathogen formerly referred to as the rosette agent [correction

The rosette agent is an obligate intracellular parasite that causes morbidity and mortality in salmonid fish. In laboratory cultures, the spore stage (2-6 microm diam.) replicates in a salmonid cell line by sequential asexual division, giving rise to daughter cells. If infected cell cultures are transferred to distilled water, the spore stage undergoes internal division to give rise to at least 5 cells each of which develops into a uniflagellated zoospore with a body of approximately 2 microm and a flagellum approximately 10 microm long. Zoosporulation does not occur in cell culture medium alone, artificial seawater, or phosphate-buffered saline. This parasite is currently classified as a member of the Class Mesomycetozoea (formerly Ichthyosporea) based on phylogenetic analyses of the small subunit ribosomal DNA of three different isolates from fish. Given these new morphological observations combined with the available molecular phylogenetic data on other mesomycetozoeans, we propose to classify the rosette agent as Sphaerothecum destruens, n. g., n. sp. This new genus has unique features including (1) intracellular development of spore stages in various organs eliciting a host granulomatous response; and (2) the differentiation of mature spores into multiple, flagellated zoospores. Taken together, these characteristics clearly distinguish it from the closely related genera Dermocystidium and Rhinosporidium.     

华中铁角蕨孢子纹饰形成的超微结构观察

利用透射电子显微镜对铁角蕨科(Aspleniaceae)华中铁角蕨(Asplenium sarelii Hook.)孢子及其纹饰的形成过程进行观察。结果表明:①华中铁角蕨孢子囊发育为薄囊蕨型;②孢子外壁表面光滑,远极面的外壁厚约0.8~1.1μm,近极面的外壁厚约1.4~1.8μm;③孢子周壁厚度约4~5μm,染色较外壁深,分为内层和外层;内层紧帖外壁表面,其上具柱状、瘤状或疣状突起;外层向外隆起形成脊状纹饰的轮廓,脊的下方具空腔,脊的顶端具翅;④铁角蕨型与鳞毛蕨型孢子外壁和周壁纹饰的形成过程具有相似性;⑤孢子的成熟度对于孢子形态的研究是至关重要的,只有完全成熟的孢子的表面纹饰才是稳定的。