Cryptic speciation and community structure of Herpotrichia juniperi, the causal agent of brown felt blight of conifers

Conifer twigs showing brown felt blight were collected along 100-m long transects at the timberline in the Swiss Alps and single-hyphal-tip cultures were prepared. Forty-seven of the sequenced 48 strains were Herpotrichia juniperi based on sequence comparisons of the internal transcribed spacers (ITS). A non-sporulating strain was tentatively identified as another, undescribed Herpotrichia species. Herpotrichia coulteri was not isolated. Most strains were from Juniperus communis var. saxatilis, the rest from Picea abies and Pinus mugo. Each twig was colonized by a different genotype as revealed by ISSR-PCR fingerprinting. More than one clone was present on some needles and twigs. Thus, importance of vegetative mycelial growth for dispersal seems to be limited to the spread of the disease to twigs of the same tree or of immediately adjacent trees, and, consequently, dispersal occurs mainly by ascospores. The H. juniperi strains could be assigned to five distinct groups based on the ISSR-PCR data. The strains from P. abies formed one of these groups but the other groups did not correlate with either host, transect or position along the transects. Multi-locus analysis based on β-tubulin, elongation factor 1-α and ITS sequences confirmed the subdivision into five groups. Population differentiation among groups was distinct with N_ST values varying between 0.545 and 0.895. H. juniperi seems to be composed of several cryptic species, one of them specific to P. abies.     

Euopsis and Harpidium,Genera of the Lichinaceae (Lichenes) with Rostrate Asci*

Based on corresponding ascocarp ontogeny and thallus structure, the genera Euopsis and Harpidium are included in the family Lichinaceae. In the two species of Euopsis, E. granatina and E. pulvinata, the apothecia develop from ascogonia in generative tissue, while in H. rutilans they are pycnoascocarps. In thallus anatomy, the species of Euopsis resemble Pyrenopsis haematopsis and allied species, while H. rutilans corresponds in structure and development of the thallus and apothecia to Pyrenopsis haemaleella (syn. P. sphinctotricha). H. rutilans is the first member of Lichinaceae known to have only a green algal symbiont. In E. granatina, two phycobionts are always present, a species of Gloeocapsa and a chlorococcalean alga. In Euopsis and Harpidium, the ascus wall is composed of an outer, non-expansible and an inner, expansible layer; the latter surrounds the protoplast as an amyloid collar, which expands during spore release into a long, tapering rostrum. In Euopsis, the outer wall layer is strongly amyloid and the upper part separated from the expanded amyloid rostrum by a non-amyloid zone, appearing like a slit in LM studies. The ultrastructure and function of the ascus in E. granatina has been studied in TEM and is interpreted as functionally unitunicate-rostrate. Unitunicate asci with short rostrum are described for P. haemaleella and P. haematopsis.     

MORPHOLOGICAL STUDIES IN PODOSPORA ANSERINA

Perithecium development in Podospora anserina begins with the formation of a coiled ascogonial initial that arises as a lateral branch from a vegetative hypha. Hyphae grow up around the initial, forming an envelope that will become the ascocarp wall. As the ascocarp increases in size, several layers of thin-walled pseudoparenchyma cells form inside the wall, especially at the apex of the ascocarp. Paraphyses arise both from the base of the ascocarp and from the innermost layer of pseudoparenchyma cells and grow inward and upward, completely filling the centrum with tightly packed filaments. During development of the ascocarp the ascogonium proliferates to form ascogenous hyphae along the base of the centrum. Asci arise from the ascogenous hyphae and grow up among the paraphyses. Meristematic growth at the ascocarp apex results in the formation of an ostiole lined with periphyses. Centrum structure in P. anserina could be interpreted as intermediate between the Xylaria and Diaporthe types.     

Freshwater ascomycetes: Jahnula apiospora (Jahnulales, Dothideomycetes), a new species from Prince Edward Island, Canada

A new ascomycete species, Jahnula apiospora (Jahnulales, Dothideomycetes), collected from submerged wood in a freshwater creek on Prince Edward Island, Canada, is described and illustrated. The characteristic features of the new species are globose to subglobose, black, ostiolate, membranous ascomata with broad, brown, subtending hyphae; a peridial wall composed of an outer layer of thick-walled cells occluded by black, amorphous material along the upper two-thirds of the ascoma; trabeculate pseudoparaphyses; cylindrical to narrowly fusoid, fissitunicate asci; and brown, one-septate, apiosporous ascospores without a gelatinous sheath or appendages.     

Convergent evolution of the lateral line system in Apogonidae (Teleostei: Percomorpha) determined from innervation

The lateral line system and its innervation were examined in two species of the family Apogonidae (Cercamia eremia [Apogoninae] and Pseudamia gelatinosa [Pseudamiinae]). Both species were characterized by numerous superficial neuromasts (SNs; total 2,717 in C. eremia; 9,650 in P. gelatinosa), including rows on the dorsal and ventral halves of the trunk, associated with one (in C. eremia) and three (in P. gelatinosa) reduced trunk canals. The pattern of SN innervation clearly demonstrated that the overall pattern of SN distribution had evolved convergently in the two species. In C. eremia, SN rows over the entire trunk were innervated by elongated branches of the dorsal longitudinal collector nerve (DLCN) anteriorly and lateral ramus posteriorly. In P. gelatinosa, the innervation pattern of the DLCN was mirrored on the ventral half of the trunk (ventral longitudinal collector nerve: VLCN). Elongated branches of the DLCN and VLCN innervated SN rows on the dorsal and ventral halves of the trunk, respectively. The reduced trunk canal(s) apparently had no direct relationship with the increase of SNs, because these branches originated deep to the lateral line scales, none innervating canal neuromast (CN) homologues on the surface of the scales. In P. gelatinosa, a CN (or an SN row: CN homologue) occurred on every other one of their small lateral line scales, while congeners (P. hayashii and P. zonata) had an SN row (CN homologue) on every one of their large lateral line scales.     

Comparative studies on the statoblasts of higher phylactolaemate bryozoans

Statoblasts of five higher phylactolaemates were compared morphologically. As a result, they were divided into two groups: Group I comprising Lophopus crystallinus, Lophopodella carteri, and Pectinatella gelatinosa, and Group II comprising Pectinatella magnifica and Cristatella mucedo. These two groups are thought to represent independent evolutionary series. In Group I and in P. magnifica, the statoblasts are curved to varying degrees after the manner of a saddle. When the dorsal and ventral valves are flattened, therefore, the contour is different between the two. In Group I, the outermost layer of a mature statoblast is hard-gelatinous and basophilic; it remains intact after the statoblast is set free. The statoblast does not float until it is dry, and the float is similar in size on both valves. In Group II, a mature statoblast is covered by a softgelatinous basophilic layer, which decays after the statoblast is released. The statoblast floats without drying, and the float is better developed on the dorsal valve than on the ventral. Moreover, in the members of Group II, large yolk granules are first formed, followed by much smaller yolk granules. When their statoblasts are treated with KOH, the shell is separated completely into two valves. These characters are common to many lower phylactolaemates. By contrast, in L. carteri and P. gelatinosa, the yolk granules are uniformly small and the capsule proper resists KOH treatment. On these points, L. crystallinus is somewhat different from these two species, suggesting its primitive nature.     

MORPHOLOGY OF NECTRIA HAEMATOCOCCA

Ascocarp development in Nectria haematoccocca begins with the formation of deeply staining coils as lateral branches of the vegetative hyphae. As these coils develop into multicellular, multi-nucleate ascogonia, they are surrounded by a pseudoparenchymatous envelope. During ascocarp development an apical meristem produces cells that elongate downward into the centrum, forming long, filamentous, apical paraphyses. When fully developed the cells of the apical paraphyses swell, producing a tissue that is pseudoparenchymatous in appearance. The ascogonium proliferates to form a layer of multinucleate ascogenous cells across the base of the ascocarp. Asci form from the ascogenous cells by means of croziers. The asci grow up among the apical paraphyses, which disintegrate as the ascocarp matures. This pattern is typical of the Nectria-type of development, indicating that this species belongs in the Hypocreales.     

A REINTERPRETATION OF THE ONTOGENY OF THE ASCOCARP OF SPECIES OF THE ERYSIPHACEAE

A study of four species of Erysiphaceae (Uncinula salicis, Podosphaera leucotricha, Erysiphe cichoracearum, and Microsphaera diffusa) revealed that the binucleate stages of the ascocarp are initiated in a similar manner to those of Diporotheca rhizophila Gordon & Shaw. The “appendages” developing on immature ascocarps are considered to be receptive hyphae. Appendages characteristic of mature ascocarps are produced much later. Lysis of certain centrum cells occurs, and asci are initiated from some of the remaining binucleate centrum cells. Resorption of centrum cells by the asci is supported by this investigation, corroborating Björling's earlier studies on Erysiphe graminis.     

Freshwater ascomycetes: <Emphasis Type="Italic">Aliquandostipite minuta</Emphasis> (Jahnulales,Dothideomycetes), a new species from Florida

An undescribed ascomycete similar to species in the Aliquandostipitaceae (Jahnulales, Dothideomycetes) was collected from submerged wood in a freshwater swamp in Big Cypress National Preserve, Florida. The characteristic features of the new species are as follows: (i) ascomata are small, sessile, light brown, globose to subglobose, papillate, and anchored to the substrate by wide, brown, septate and subtending hyphae; peridial wall is composed of 1 to 2 layers of large, angular cells with large lumens; (ii) asci are ovoid to broadly clavate, and fissitunicate; (iii) ascospores are one-septate, fusiform, multiguttulate, pale brown, surrounded by a fusiform gelatinous sheath, and equipped with numerous filamentous appendages around the midseptum. The new fungus is most similar to Aliquandostipite crystallinus, from which it differs in overall smaller size and morphology. This new fungus is described and illustrated herein as A. minuta.     

MORPHOLOGY OF HYPOMYCES TRICHOTHECOIDES

Large, spirally coiled initials embedded in a subiculum develop into multicellular, multinucleate ascogonia. Hyphae grow up around them to form a prosenchymatous perithecial wall. The ascogonia give rise to multinucleate ascogenous cells from which croziers and asci form. As the ascocarp develops, an apical meristem produces uninucleate cells that elongate downward into long, slender filaments, the apical paraphyses. From a basal layer of ascogenous cells, asci grow up among the apical paraphyses, which disintegrate as the ascocarp matures. Ascospores are verrucose, with obtuse apiculi. This pattern of development is typical of the Nectria-type of Luttrell.     

Phylogenetic affiliation of the desert truffles <Emphasis Type="Italic">Picoa juniperi</Emphasis> and <Emphasis Type="Italic">Picoa lefebvrei</Emphasis>

The molecular phylogeny and comparative morphological studies reported here provide evidence for the recognition of the genus Picoa, an hypogeous desert truffle, in the family Pyronemataceae (Ascomycota, Pezizales). Picoa juniperi and Picoa lefebvrei were reassigned to the genus Picoa based on large subunit (LSU) sequence (28S) rDNA and internal transcribed spacer (ITS) rDNA (including the partial 18S, ITS1, ITS2, 5.8S gene, and partial 28S of the nuclear rDNA) data. Morphological studies of spores, asci, perida, and gleba revealed high similarities between P. lefebvrei and P. juniperi, thereby confirming the membership of both species in the genus Picoa. These two species were primarily distinguishable based on ascospore ornamentation.     

狗脊孢壁发育超微结构的研究

鳞毛蕨型孢子类型众多,初步研究表明形态相似的孢子类型其孢壁发育特征存在差异,因此有必要对各代表类群的孢壁发育进行深入地研究。该文利用透射电镜对乌毛蕨科(Blechnaceae)狗脊(Woodwardia japonica)孢壁结构和发育的超微结构进行研究。结果表明:(1)狗脊孢子囊的结构由外向内分别为孢子囊壁细胞、两层绒毡层细胞和孢子母细胞;(2)狗脊孢子具乌毛蕨型(Blechnoid type)外壁,表面光滑,由两层构成,裂缝区域具辐射状的槽;(3)周壁属于空心型(cavity type),由四层构成,从内向外分别为P1、P2、P3和P4层,前三层叠合在一起,层间有不同程度的空隙,P4层与前三层之间具有明显而连续的空腔,并隆起形成片状褶皱纹饰;(4)有小球体和小杆共同参与孢子周壁的形成,周壁部分或全部来源于孢子囊壁细胞。综上所述,狗脊孢子与同属于鳞毛蕨型的贯众(Cyrtomium fortunei)和朝鲜介蕨(Dryoathyrium coreanum)孢壁的发育在周壁结构、周壁各层的发育顺序、周壁来源和参与成壁的特征物质等方面存在差异。该研究有利于进一步理解蕨类植物孢壁所蕴含的分类和演化上的科学意义和价值。     

ULTRASTRUCTURE OF ASCOCARP DEVELOPMENT IN SPORORMIA AUSTRALIS

Thin sections taken from intact ascocarps were examined to trace the developmental sequence of ascocarp formation in Sporormia australis Speg. The ascocarp originated from a uninucleate vegetative hyphal cell which underwent repeated divisions and formed an ascostroma. In the center of the young ascostroma a cavity formed, apparently from cell disintegrations, and enlarged as the ascocarp enlarged. Within the cavity pseudoparaphyses developed from undifferentiated pseudoparenchymatous cells at the apex of the cavity and extended downward. Ascogenous hyphae arose from proliferating uninucleate cells at the base of the cavity. As the ascocarp matured, the pseudoparenchymatous cells differentiated into three layers, none of which were considered homologous to the perithecial wall lining the cavity of pyrenomycetes. The cells of the apex were not differentiated into layers and light microscopy revealed the presence of an ostiole through which bitunicate asci discharged their eight 4-celled ascospores.     

THE CENTRUM OF THE SOOTY MOLD ASCOMYCETE LIMACINULA SAMOENSIS

Centrum development in the sooty mold Ascomycete Limacinula samoensis von Hoehnel emend. Reynolds proceeds in an ascostroma which begins as a small cushion of somatic tissue and enlarges by multiplication of cells in an apical region and by cell enlargement. A two-layered ascocarp wall initially surrounds a pseudoparenchymatous core into which the bitunicate asci protrude. Interascal strands of pseudoparenchymatous tissue disintegrate at maturity of the ascocarp. An apical meristem eventually culminates activity with formation of a short ostiolate neck. Centrum development is homologous to the Dothidea type. The centrum development of other capnodiaceous fungi is reviewed.     

A LIGHT- AND ELECTRON-MICROSCOPIC SURVEY OF ALGAL CELL WALLS. I. PHAEOPHYTA AND RHODOPHYTA

Dawes , Clinton J., Flora M. Scott , and E. Bowler . (U. California, Los Angeles.) A light- and electron-microscopic survey of algal cell walls. I. Phaeophyta and Rhodophyta. Amer. Jour. Bot. 48(10): 925–934. Illus. 1961.—An introductory survey of the structure of the cell walls of brown, red, and green algae, as seen under light and electron microscopes, has been completed. In the present paper (Part I) the structure of the thalli of the Phaeophyta and Rhodophyta is compared, and the occurrence of intercellular spaces, pitting, and microfibrillar patterns is discussed. A detailed comparison of the cell-wall structure and growth of a brown alga, Dictyota flabellata, and of a red alga, Helminthocladia californica, is also presented. In Dictyota, typical of the brown algae, the microfibrillar pattern in the apical cells and in the adjacent cells of the thallus tip is reticulate. In mature cells, the microfibrils are dominantly parallel in orientation. Pits, which are fields of closely set pores, are distinctive. The microfibrils in the pit areas are masked by non-fibrillar material. Helminthocladia, with a cell wall characteristic of the red algae, differs from Dictyota in that the microfibrillar pattern is reticulate at all ages of the cell and throughout the thallus. In the pit areas, the microfibrils are not masked by amorphous material. Pit connections, characteristic of the Florideae, can be divided into 2 major groups. Either the pit connection is an open channel between 2 adjacent cells, or it is composed of numerous plasmodesmata traversing a continuous, loose, microfibrillar wall. The techniques of the survey are emphasized in that fragmented cell walls were studied, and, also, chemically cleared material was constantly compared with fresh material under light and electron microscopes. It is concluded that the cell wall, as a taxonomic character, is of value only in delimiting the Phaeophyta and Rhodophyta.     

Species-specific ITS primers for the identification of Picoa juniperi and Picoa lefebvrei and using nested-PCR for detection of P. juniperi in planta

Desert truffles, hypogeous Pezizales (Ascomycota), are difficult to identify due to evolutionary convergence of morphological characters among taxa that share a similar habitat and mode of spore dispersal. Also, during their symbiotic phase, these are barely distinguishable morphologically, and molecular probes are needed for their identification. We have developed a PCR-based method for the identification of Picoa juniperi and Picoa lefebvrei based on internal transcribed spacers of rDNA. Two PCR primers specific for P. lefebvrei (FLE/RLE) and two specific for P. juniperi (FJU/RJU) were designed. A collection of samples from different geographical areas representing diversity of these species were examined for unique regions of internal transcribed spacers 1, 2 and 5.8S gene of rDNA (ITS) compared to other closely related species. Annealing temperatures and extension times were optimized for each set of primers for maximum specificity and efficiency. They proved to be efficient to specifically detect the presence of P. juniperi and P. lefebvrei by PCR and neither set amplified purified DNA from other truffle species as well as some ascomycetous fungi. The partial small subunit of ribosomal DNA genes of P. juniperi were amplified with the genomic DNA extracted from Helianthemum ledifolium var. ledifolium roots by nested polymerase chain reaction (PCR) using the universal fungal primer pair ITS1/ITS4 and specific primer pair FTC/RTC, which was designed based on internal transcribed spacer 1, 2 and 5.8S gene of rDNA sequences of P juniperi. The nested-PCR was sensitive enough to re-amplify the direct-PCR product, resulting in a DNA fragment of 426 bp. The efficacy of nested-PCR showed that it could re-amplify the direct-PCR product and detect 200 fg genomic DNA.     

Four ectomycorrhizae of Pyronemataceae (Pezizomycetes) on Chinese Pine (<Emphasis Type="Italic">Pinus tabulaeformis</Emphasis>): morpho-anatomical and molecular-phylogenetic analyses

Morphological and anatomical characters of four ectomycorrhizae with affinities to the genera Humaria, Geopora, and Trichophaea of Pyronemataceae (Pezizomycetes, Ascomycota) on Chinese Pine (Pinus tabulaeformis) are described. The ectomycorrhizae are yellowish brown to brown, and have pseudoparenchymatous outer mantle layers and partially warty emanating hyphae with thick walls and without clamps. Intrahyphal hyphae are present, and no rhizomorphs are formed. The four ectomycorrhizae are distinguishable by differences in cell shape of outer mantle layers and the presence of cystidia. Ectomycorrhizae of a possible Humaria species (Pinirhiza humarioides) lack cystidia and have irregularly inflated cells on the outer mantle layer that are connected with thin septa. The two ectomycorrhizae showing probable affinities to Geopora species (“P. daqingensis” and “P. geoporoides”) possess row-like arranged cells in the outer mantle layer and cell heaps, and differ by the presence or absence of cystidia as well as by the structure of the inner mantle layers. Ectomycorrhizae likely having been formed by a Trichophaea species (“P. trichophaeoides”) have oval to polygonal cells and no cystidia. The possible taxa affiliations were assessed by molecular-phylogenetic analyses of the internal transcribed spacer (ITS) and partial large subunit (LSU) nrDNA. Morphological and anatomical characters are discussed against the background of the LSU phylogeny.     

Utilization of immobilized benthic algal species for N and P removal

Laboratory experiments were performed to study the growth rate and phosphorus (P) and nitrogen (N) uptakes of eight benthic microalgae species isolated from different sources of pig manure. Cells immobilized in calcium alginate beads were cultured with three replicates for each species. P removal rates obtained for the unicellular self-aggregating benthic species (Palmellopsis gelatinosa, Chlorosarcinopsis sp., and Macrochloris sp.) were markedly higher than those obtained in previous published experiments. N removal rates were highest for Macrochloris sp., Chlorosarcinopsis sp., and Euglena sp. 2 and comparable to the maximum rates obtained by other authors. Our results show an excellent efficiency of autochthonous benthic species for nutrient removal, especially for P, and call attention to their use for wastewater treatment.     

ASCOCARPIC CENTRUM ONTOGENY OF SPECIES OF HYPODERMATACEAE OF CONIFERS. II

Ascocarpic studies of the ontogeny of Lophodermium nitens disclosed a type of development unlike that of all other species of Hypodermataceae occurring on conifer needles. For this reason the centrum of L. nitens is designated as Type III and is compared with Type I (Gordon, 1966). Because L. nitens produces its ascocarp in several tissues of various species of pine, the ontogeny of ascocarps in different locations is discussed and illustrated. The most significant ontogenetic feature of the ascocarp of L. nitens is a layer of hyaline cells in the primordium; this layer is meristematic and gives rise to all subsequent structures of the centrum.