Minutisphaerales (Dothideomycetes, Ascomycota): a new order of freshwater ascomycetes including a new family, Minutisphaeraceae, and two new species from North Carolina, USA.

Minutisphaera is a recently established genus of freshwater Dothideomycetes characterized by small, globose to subglobose or apothecioid, erumpent to superficial, brown ascomata; fissitunicate, eight-spored, ovoid to obclavate asci; and 1-2-septate, clavate to broadly fusiform, hyaline to pale brown ascospores with or without a gelatinous sheath and filamentous appendages. The genus currently contains two species: M. fimbriatispora, the type species, and M. japonica. The higher-level phylogenetic relationship of Minutisphaera within the Dothideomycetes currently is unresolved. To establish the phylogenetic position of Minutisphaera within the Dothideomycetes and evaluate the phylogenetic affinities of newly collected Minutisphaera-like taxa, we sequenced three rDNA regions-18S, ITS1-5.8SITS2 (ITS) and 28S nuc rDNA, and a protein-coding gene, MCM7, for newly collected strains of Minutisphaera. Based on maximum likelihood and Bayesian analyses of a combined dataset (18S and 28S) composed of 167 taxa, a more refined dataset (28S and MCM7) comprising 52 taxa and a separate ITS dataset, and an examination of morphology, we describe and illustrate two new species of Minutisphaera. The Minutisphaera clade was strongly supported within the Dothideomycetes with likelihood and Bayesian statistics but did not share phylogenetic affinities with any existing taxonomic group within the Dothideomycetes. We therefore establish a new order, Minutisphaerales, and new family, Minutisphaeraceae, for this monophyletic clade of freshwater ascomycetes. Chemical analysis of the organic extract M. aspera (G427) resulted in isolation and characterization of five known secondary metabolites, of which four were dipeptides (1-4) and one an aromatic polyketide (5). Conversely, two aromatic polyketides (5, 6) were isolated and identified from the organic extract of M. parafimbriatispora (G156-4). The isolated compounds were tested for their antimicrobial activity against an array of bacteria and fungi. Compound 6 showed promising activity against Staphylococcus aureus and Mycobacterium smegmatis with minimal inhibitory concentration values of 30 and 60 µg/mL, respectively.

Phylogenetic Diversity of Sponge-Associated Fungi from the Caribbean and the Pacific of Panama and Their In Vitro Effect on Angiotensin and Endothelin Receptors.

Fungi occupy an important ecological niche in the marine environment, and marine fungi possess an immense biotechnological potential. This study documents the fungal diversity associated with 39 species of sponges and determines their potential to produce secondary metabolites capable of interacting with mammalian G-protein-coupled receptors involved in blood pressure regulation. Total genomic DNA was extracted from 563 representative fungal strains obtained from marine sponges collected by SCUBA from the Caribbean and the Pacific regions of Panama. A total of 194 operational taxonomic units were found with 58% represented by singletons based on the internal transcribed spacer (ITS) and partial large subunit (LSU) rDNA regions. Marine sponges were highly dominated by Ascomycota fungi (95.6%) and represented by two major classes, Sordariomycetes and Dothideomycetes. Rarefaction curves showed no saturation, indicating that further efforts are needed to reveal the entire diversity at this site. Several unique clades were found during phylogenetic analysis with the highest diversity of unique clades in the order Pleosporales. From the 65 cultures tested to determine their in vitro effect on angiotensin and endothelin receptors, the extracts of Fusarium sp. and Phoma sp. blocked the activation of these receptors by more than 50% of the control and seven others inhibited between 30 and 45%. Our results indicate that marine sponges from Panama are a "hot spot" of fungal diversity as well as a rich resource for capturing, cataloguing, and assessing the pharmacological potential of substances present in previously undiscovered fungi associated with marine sponges.

Phylogeny and morphology of dematiaceous freshwater microfungi from Perú.

A survey of freshwater ascomycetes conducted along an elevational gradient in Perú in the Districts of Cusco, Junín, and Madre de Dios yielded specimens of Cancellidium applanatum, Cordana abramovii, Sporoschisma juvenile, S. uniseptatum, and S. saccardoi. With the exception of S. saccardoi, these are new records for Perú. Molecular data was generated for three previously unsequenced species: Cancellidium applanatum, Cordana abramovii and Sporoschisma saccardoi. These taxa are reported herein from the neotropics with an accompanying phylogeny based on partial 28S nuclear ribosomal large-subunit sequence data. The sexual morph of S. saccardoi has previously been linked to Melanochaeta hemipsila through cultural studies. Molecular data from ascospores and conidia of M. hemipsila and S. saccardoi, respectively, were used to demonstrate a genetic connection of the sexual and asexual morphs of these fungi for the first time, resulting in the new combination Sporoschisma hemipsila being made.

Freshwater ascomycetes: Minutisphaera (Dothideomycetes) revisited, including one new species from Japan.

During investigations of freshwater ascomycetes we found one interesting taxon from Aomori (Japan), as well as three additional taxa from North Carolina (USA), which were morphologically similar to Minutisphaera, a recently described freshwater fungus in the Dothideomycetes. The ascomata of all the collections bore dark hair-like structures around the ostiolar region, obovoid to obclavate bitunicate asci, and one to three septate hyaline to brown ascospores with a sheath (in material from Japan), and with both sheath and appendages (in material from the USA). The apothecial ascomata of these taxa, however, differ from those of the type species of the genus, which are perithecial. Two collections of Minutisphaera-like fungi from the USA were morphologically quite similar but differed in ascospore size. To assess the phylogenetic affinities of Minutisphaera-like taxa with the type species, M. fimbriatispora, we sequenced 18S and 28S nrDNA of five newly collected strains of Minutisphaera. We also sequenced the nrDNA for the entire internal transcribed spacer region of 10 strains to assess interspecific and intraspecific variation with M. fimbriatispora. Additionally we examined the secondary metabolite profiles of two strains from USA. Based on maximum likelihood and Bayesian analyses of combined 18S and 28S, and separate ITS sequences, as well as examination of morphology, we describe and illustrate a new species, M. japonica. One collection from North Carolina is confirmed as M. fimbriatispora, while two other collections are Minutisphaera-like fungi that had a number of similar diagnostic morphological characters but differed only slightly in ascospore sizes. The phylogeny inferred from the internal transcribed spacer region suggested that two out of the three North Carolina collections may be novel and perhaps cryptic species within Minutisphaera. Organic extracts of Minutisphaera from USA, M. fimbriatispora (G155-1) and Minutisphaera-like taxon (G156-1), revealed the presence of palmitic acid and (E)-hexadec-9-en-1-ol as major chemical constituents. We discuss the placement of the Minutisphaera clade within the Dothideomycetes. The description of the genus Minutisphaera is emended to accommodate M. japonica within Minutisphaera.

New species and new records of freshwater ascomycetes from Brazil and Costa Rica.

During independent surveys for freshwater ascomycetes in Brazil and Costa Rica, two new species, Torrentispora pilosa and Vertexicola ascoliberatus, and nine previously described species were recovered. Among the described species, Annulatascus biatriisporus, Anthostomella aquatica, Tamsiniella labiosa and Torrentispora crassiparietis are reported for the first time from the western hemisphere, Aniptodera chesapeakensis, Chaetosphaeria lignomollis and Jahnula seychellensis are new records for South America and Annulatascus velatisporus and Ophioceras venezuelensis are reported for the first time for Brazil. The description of the genus Torrentispora is emended to accommodate T. pilosa. The new species are described and illustrated and a brief description is provided for all new records.

Three new genera representing novel lineages of Sordariomycetidae (Sordariomycetes, Ascomycota) from tropical freshwater habitats in Costa Rica.

Three new genera are established in the Sordariomycetidae based on morphological and molecular data (SSU and LSU nrDNA) to accommodate five ascomycete species collected from submerged woody debris in freshwater habitats from Costa Rica. The genus Bullimyces contains three new species, B. communis, B. costaricensis and B. aurisporus. Bullimyces is characterized by globose to subglobose, membranous, black, ostiolate ascomata; deliquescent, hyaline, globose cells that fill the center of the centrum; unitunicate asci that deliquesce early in some species; and septate, thick-walled ascospores with or without gelatinous sheaths or appendages. Bullimyces species form a well supported clade with 100% bootstrap support, but the position of the genus in the Sordariomycetidae remains unclear. The second genus, Riomyces, is represented by a single species, R. rotundus. Riomyces is characterized by globose to subglobose, membranous, black, ostiolate ascomata, unitunicate, cylindrical asci, hyaline, globose cells that fill the hamathecium and septate, thick-walled ascospores with a gelatinous sheath. Although Riomyces is morphologically similar to Bullimyces, the two genera did not group together with support in any analysis. The third genus, Hydromelitis, is represented by a single species, H. pulchella. Hydromelitis is characterized by pyriform, membranous, black, ostiolate ascomata, unitunicate asci lacking an apical structure, simple, thin-walled, septate paraphyses and hyaline to golden yellow, multiseptate, thick-walled ascospores with a gelatinous sheath. Bullimyces, Riomyces and Hydromelitis were nested within an unsupported clade consisting of members of the Ophiostomatales, Magnaporthales and freshwater Annulatacaceae sensu lato and sensu stricto.

Freshwater ascomycetes: Natipusillaceae, a new family of tropical fungi, including Natipusilla bellaspora sp. nov. from the Peruvian Amazon.

A new ascomycete species, Natipusilla bellaspora, collected from submerged woody debris in a freshwater stream at Los Amigos Biological Station, Madre De Dios in the Peruvian Amazon is described and illustrated. This fungus is characterized by small, globose to subglobose, hyaline ascomata; small, globose to subglobose, eight-spored fissitunicate asci; one-septate, multiguttulate ascospores with two different gelatinous sheaths, an outer amorphous sheath that enlarges in water and an inner sheath that has a distinctive persistent shape and is attached to the ascospore apex. Morphologically N. bellaspora differs from other Natipusilla species in having larger ascospores and two ascospore sheaths. A second Natipusilla species, N. limonensis, is reported for the first time from Peru. Based on the unique morphological characters of taxa in Natipusilla and results of previous molecular phylogenetic analyses with other members of the Dothideomycetes, we establish Natipusillaceae fam. nov. for this unique tropical freshwater clade.

Freshwater ascomycetes: two new species of Lindgomyces (Lindgomycetaceae, Pleosporales, Dothideomycetes) from Japan and USA.

During independent surveys of freshwater ascomycetes in Japan and USA two new species of Lindgomyces were collected from submerged wood in freshwater. These species are described and illustrated based on morphological data and phylogenetic relationships based on analyses of nuclear ribosomal sequence data (partial SSU and LSU, and ITS). Lindgomyces apiculatus, collected in Japan, is characterized by immersed to erumpent, globose to subglobose ascomata; fissitunicate, cylindrical to clavate asci; and fusiform, one-septate ascospores with acute ends and short terminal appendages. Lindgomyces lemonweirensis, collected in Wisconsin, USA, differs from L. apiculatus in having clavate to cymbiform asci and oblong to fusiform ascospores that are distinctively multiguttulate and surrounded by an oval, ephemeral gelatinous sheath. The new species formed a strongly supported clade within the family Lindgomycetaceae (Pleosporales, Dothideomycetes) based on analyses of combined SSU and LSU sequence data. In addition phylogenetic analyses with ITS sequence data support the establishment of the new taxa as separate species within Lindgomyces because they were separated from each other and other Lindgomyces species based on maximum likelihood bootstrap and Bayesian analyses.

Minutisphaera and Natipusilla: two new genera of freshwater Dothideomycetes.

Two new genera are established in the Dothideomycetes based on morphological and molecular data (SSU and LSU nuclear ribosomal sequences) to accommodate four ascomycete species collected from woody debris submerged in freshwater habitats. The genus Minutisphaera is represented by a single species, M. fimbriatispora, which was collected from freshwater habitats in temperate forests in North America. It has small, superficial, brown, subglobose, papillate pseudothecia with dark, irregularly twisted hairs around the papillae, fissitunicate asci, septate pseudoparaphyses, and hyaline, one-septate ascospores surrounded by a gelatinous sheath and having spine-like appendages radiating around the ascospore at the septum. Minutisphaera formed a strongly supported clade with Farlowiella carmichaeliana. The second genus, Natipusilla, contains three new species, N. decorospora, N. limonensis and N. naponensis, which were collected from Central and South America. Natipusilla is characterized by small, superficial, light-colored, globose pseudothecia, fissitunicate asci, few or no pseudoparaphyses, and hyaline, one-septate to tardily two- or three-septate ascospores with or without a gelatinous sheath. The three Natipusilla species form a well supported clade, but their relationship to other members of the Dothideomycetes remains unclear.

Population genetics of the aquatic fungus Tetracladium marchalianum over space and time.

Aquatic hyphomycete fungi are fundamental mediators of energy flow and nutrient spiraling in rivers. These microscopic fungi are primarily dispersed in river currents, undergo substantial annual fluctuations in abundance, and reproduce either predominantly or exclusively asexually. These aspects of aquatic hyphomycete biology are expected to influence levels and distributions of genetic diversity over both spatial and temporal scales. In this study, we investigated the spatiotemporal distribution of genotypic diversity in the representative aquatic hyphomycete Tetracladium marchalianum. We sampled populations of this fungus from seven sites, three sites each in two rivers in Illinois, USA, and one site in a Wisconsin river, USA, and repeatedly sampled one population over two years to track population genetic parameters through two seasonal cycles. The resulting fungal isolates (N = 391) were genotyped at eight polymorphic microsatellite loci. In spite of seasonal reductions in the abundance of this species, genotypic diversity was consistently very high and allele frequencies remarkably stable over time. Likewise, genotypic diversity was very high at all sites. Genetic differentiation was only observed between the most distant rivers (∼450 km). Clear evidence that T. marchalianum reproduces sexually in nature was not observed. Additionally, we used phylogenetic analysis of partial ß-tubulin gene sequences to confirm that the fungal isolates studied here represent a single species. These results suggest that populations of T. marchalianum may be very large and highly connected at local scales. We speculate that large population sizes and colonization of alternate substrates in both terrestrial and aquatic environments may effectively buffer the aquatic populations from in-stream population fluctuations and facilitate stability in allele frequencies over time. These data also suggest that overland dispersal is more important for structuring populations of T. marchalianum over geographic scales than expected.

A molecular phylogenetic assessment of Massarina ingoldiana sensu lato.

Massarina ingoldiana occurs worldwide on a variety of dead plant substrates in aquatic habitats. This species has been accommodated in Massarina or Lophiostoma in Pleosporales, Dothideomycetes, but the validity of either of these taxonomic placements has not been confirmed with molecular data. In addition morphological variations occur among different populations of this species causing problems in identification. To evaluate the generic placement and monophyly of M. ingoldiana and the taxonomic usefulness of variable morphological features, phylogenetic analyses based on SSU and LSU sequences of ribosomal DNA were conducted for 10 putative strains of this species and its relatives. Phylogenies revealed that M. ingoldiana sensu lato is polyphyletic and comprises two distinct lineages within Pleosporales. Neither lineage was congeneric with either Massarina or Lophiostoma. Based on molecular data and a reevaluation of morphology, two new genera, Lindgomyces and Tingoldiago, are established for the two lineages of M. ingoldiana sensu lato. Lindgomyces includes four species, L. ingoldianus comb. nov. (= M. ingoldiana sensu stricto), L. rotundatus sp. nov. (= M. ingoldiana sensu lato), L. cinctosporae sp. nov. and L. breviappendiculatus comb. nov. (= Lophiostoma breviappendiculatum). A new aquatic family, Lindgomycetaceae, is proposed for Lindgomyces and its sister taxon, Massariosphaeria typhicola. Isolates of a fungus from submerged Phragmites, with ascospores similar to those of M. ingoldiana, occurred in an additional single species lineage distant from that of M. ingoldiana (Lindgomyces). This fungus is described as Tingoldiago graminicola gen. & sp. nov. The discovery that Tingoldiago, which occurs in a lineage distantly related to Lindgomyces but has morphologically similar ascospores and ascospore sheaths, suggests that the elaborate ascospore sheath in M. ingoldiana has arisen in two separate lineages as a result of convergent evolution in response to the aquatic environment. The large gelatinous sheath previously was considered one of the most distinctive and stable features for species identification of M. ingoldiana.

Freshwater ascomycetes: Alascospora evergladensis, a new genus and species from the Florida Everglades.

Alascospora evergladensis, a freshwater ascomycete collected from submerged dead petioles of Nymphaea odorata during a survey of aquatic fungi along a phosphorus gradient in the Florida Everglades, is described and illustrated as a new genus and species in the Pleosporales (Pleosporomycetidae, Dothideomycetes). The new fungus is unique among genera in the Pleosporales based on a combination of morphological characters that include light brown, translucent, membranous, ostiolate ascomata with dark, amorphous material irregularly deposited on the peridium, especially around the ostiole; globose, fissitunicate, thick-walled asci; septate pseudoparaphyses; and 1-septate ascospores that are hyaline when young, and surrounded by a hyaline gelatinous sheath that is wing-shaped in outline on each side of the ascospore. The sheath is distinctive in that it first expands in water and is translucent, then condenses and darkens around older ascospores, giving them a dark brown, verruculose appearance.

Lucidascocarpa pulchella, a new ascomycete genus and species from freshwater habitats in the American tropics.

A new fungus collected from submerged wood in Costa Rica and Ecuador has ascostromatic ascomata with fissitunicate asci and lacks pseudoparaphyses, characters that place it in the Dothideaceae (Dothideales). It is unusual in the order because it has white ascomata. Based on other morphological characters however this fungus could not be accommodated in any existing genus in the Dothideaceae and it is described herein as a new genus and species, Lucidascocarpa pulchella. These morphological features are characteristic of L. pulchella: ascomata glistening, white, each with a long, periphysate neck; a membranous peridium composed of 5-7 thin-walled, hyaline cells; pseudoparaphyses absent; asci fissitunicate, clavate, eight-spored; ascospores seven-septate, hyaline, multiguttulate, verruculose, surrounded by a large, regular, gelatinous sheath.

Freshwater ascomycetes: new and noteworthy species from aquatic habitats in Florida.

As part of a distributional study of freshwater ascomycetes in Florida, a number of new taxa were encountered. The new taxa include six Sordariomycetes, Aniptodera megaloascocarpa sp. nov., Flammispora pulchra sp. nov., Hanliniomyces hyaloapicalis gen. et sp. nov., Lockerbia striata sp. nov., Phomatospora triseptata sp. nov. and Physalospora limnetica sp. nov., and three Dothideomycetes, Caryospora obclavata sp. nov., Lepidopterella tangerina sp. nov. and Ophiobolus shoemakeri sp. nov. These taxa are described and illustrated. Six additional species are reported from Florida for the first time; among them, two species are new reports from freshwater habitats.

Megalohypha, a new genus in the Jahnulales from aquatic habitats in the tropics.

A new fungus collected from submerged wood in tropical forest streams in Panama and Thailand is described as a new genus and species in the Jahnulales. This fungus, Megalohypha aqua-dulces, is described based on ascospore morphology, which differs substantially from that of the other genera in the Jahnulales. It has these morphological features: ascomata hyaline, translucent, with subtending, wide, brown, septate, stoloniferous hyphae; peridium of large, thin-walled cells; hamathecium of septate pseudoparaphyses; asci clavate, fissitunicate, 8-spored; and ascospores 1-septate, brown, rough-walled, with longitudinal sulcate striations. An interesting feature of Megalohypha aqua-dulces, which it shares with some other taxa in Jahnulales, is the presence of both sessile and stalked fruiting bodies. In addition, the stalks have morphology that consists of a wide hypha with a narrower hypha apparently enclosed within and the stalks are strongly constricted at the septa.

Three new species of Luttrellia from temperate and tropical freshwater habitats.

Three pyrenomycetes collected from woody debris submerged in freshwater habitats are described as new species of Luttrellia: L. guttulata sp. nov., L. halonata sp. nov. and L. paroulospora sp. nov. A collection of L. estuarina, the type species of the genus, having ascospores surrounded by a gelatinous sheath, a characteristic not observed in the type specimen, is reported. The genus description is emended to include fungi with 4- or 8-spored asci and ascospores with or without a gelatinous sheath. Luttrellia is distinguished from other genera in the Halosphaeriaceae by hyaline, phragmoseptate, and thick-walled ascospores with or without a gelatinous sheath.

Decaspirones A-E, bioactive spirodioxynaphthalenes from the freshwater aquatic fungus Decaisnella thyridioides.

Decaspirones A-E (1-5), five new compounds related to the palmarumycins, were isolated from cultures of the freshwater aquatic fungal species Decaisnella thyridioides. The known compound palmarumycin CP1 (6) was also obtained. The structures of 1-5 were determined by analysis of NMR and MS data, and their relative configurations were assigned by analysis of 1H NMR J-values and NOESY data. The structure of the lead compound 1 was confirmed by X-ray crystallographic analysis. Compounds 1-5 possess a trans ring fusion not previously reported in members of this structural class. The absolute configuration of 1 was assigned using the modified Mosher method. Compounds 1-5 showed potent antifungal and antibacterial activity.

Heliconols A-C: antimicrobial hemiketals from the freshwater aquatic fungus Helicodendron giganteum.

[Structure: see text] Cultures of the freshwater aquatic fungus Helicodendron giganteum afforded three new compounds, heliconols A-C (1-3), that contain an unusual reduced furanocyclopentane unit. The structures of these metabolites were assigned by analysis of 1D and 2D NMR data. The absolute configuration of heliconol A (1) was assigned by single-crystal X-ray crystallographic analysis of its dibromobenzoate derivative. Heliconol A showed antifungal and antibacterial activities in disk diffusion assays.

Altenuene derivatives from an unidentified freshwater fungus in the family Tubeufiaceae.

Four new altenuene derivatives called dihydroaltenuenes A (1) and B (2) and dehydroaltenuenes A (3) and B (4), along with five known compounds, including isoaltenuene (5), altenuene (6), and 5'-epialtenuene (7), were isolated from cultures of an unidentified freshwater aquatic fungal species in the family Tubeufiaceae. The structures of 1-4 were determined by analysis of NMR and MS data. The relative stereochemistry was determined on the basis of (1)H NMR J-values and NOE data, while the absolute configuration of a representative member of the group (5) was assigned by CD spectral analysis of its bis-N,N-dimethylaminobenzoate derivative. Compounds 1, 3, 4, 5, and 6 showed antibiotic activity against Gram-positive bacteria.

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists.

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.