The Life Cycle of Didymium laxifilum and Physarum album on Oat Agar Culture

The plasmodial slime molds is the largest group in the phylum Amoebozoa. Its life cycle includes the plasmodial trophic stage and the spore‐bearing fruiting bodies. However, only a few species have their complete life cycle known in details so far. This study is the first reporting the morphogenesis of Didymium laxifilum and Physarum album. Spores, from field‐collected sporangia, were incubated into hanging drop cultures for viewing germination and axenic oat agar plates for viewing plasmodial development and sporulation. The spores of D. laxifilum and P. album germinated by method of V‐shape split and minute pore, respectively. The amoeboflagellates, released from spores, were observed in water film. The phaneroplasmodia of two species developed into a number of sporangia by subhypothallic type on oat agar culture. The main interspecific difference of morphogenesis was also discussed.     

PRELIMINARY INVESTIGATION OF THE DOUBLE-DIFFUSION TECHNIQUE AS A TOOL IN DETERMINING RELATIONSHIPS AMONG SOME MYXOMYCETES,ORDER PHYSARALES

Double-diffusion technique was used to investigate myxomycete relationships within the order Physarales. Extracts of plasmodia of 22 slime mold isolates were reacted with five antisera produced to Plasmodia of Didymium nigripes, Physarella oblonga, Physarum polycephalum, Physarum gyrosum and Fuligo septica. Two isolates of Fuligo septica tested alike. Four isolates of Physarum pusillum did not test alike, and no valid conclusion of the relationship of this species was possible. These isolates showed strong serological affinity: (1) Physarum gyrosum, Physarella oblonga, two isolates of Fuligo septica, and possibly two isolates of Physarum pusillum, and Physarum tenerum; (2) Physarum polycephalum and Physarum flavicomum; (3) Fuligo septica and many of the species tested; (4) Didymium nigripes and at least one isolate of Didymium iridis. In most cases serologial relationships among species tested did not coincide with current taxonomy based on morphology of fructification.     

Reclassification of Two <Emphasis Type="Italic">Peronospora</Emphasis> Species Parasitic on <Emphasis Type="Italic">Draba</Emphasis> in <Emphasis Type="Italic">Hyaloperonospora</Emphasis> Based on Morphological and Molecular Phylogenetic Data

On the family Brassicaceae, the causal agent responsible for downy mildew disease was originally regarded as a single species, Peronospora parasitica (now under Hyaloperonospora), but it was recently reconsidered to consist of many distinct species. In this study, 11 specimens of Peronospora drabae and P. norvegica parasitic on the genus Draba were investigated morphologically and molecularly. Pronounced differences in conidial sizes (P. drabae: 14–20 × 12.5–15.5 μm; P. norvegica: 20–29 × 15.5–22 μm) and 7.8% sequence distance between their ITS1-5.8S-ITS2 rDNA sequences confirmed their status as distinct species. Based on ITS phylogeny and morphology (monopodially branching conidiophores, flexuous to sigmoid ultimate branchlets, hyaline conidia and lobate haustoria), the two species unequivocally belong to the genus Hyaloperonospora and not to Peronospora to which they were previously assigned. Therefore, two new combinations, Hyaloperonospora drabae and H. norvegica, are proposed. The two taxa are illustrated and compared using the type specimen for H. norvegica and authentic specimens for H. drabae, which is lectotypified.     

Genetic structure of the protist Physarum albescens (Amoebozoa) revealed by multiple markers and genotyping by sequencing

Myxomycetes are terrestrial protists with many presumably cosmopolitan species dispersing via airborne spores. A truly cosmopolitan species would suffer from outbreeding depression hampering local adaptation, while locally adapted species with limited distribution would be at a higher risk of extinction in changing environments. Here, we investigate intraspecific genetic diversity and phylogeography of Physarum albescens over the entire Northern Hemisphere. We sequenced 324 field collections of fruit bodies for 1–3 genetic markers (SSU, EF1A, COI) and analysed 98 specimens with genotyping by sequencing. The structure of the three-gene phylogeny, SNP-based phylogeny, phylogenetic networks, and the observed recombination pattern of three independently inherited gene markers can be best explained by the presence of at least 18 reproductively isolated groups, which can be seen as cryptic species. In all intensively sampled regions and in many localities, members of several phylogroups coexisted. Some phylogroups were found to be abundant in only one region and completely absent in other well-studied regions, and thus may represent regional endemics. Our results demonstrate that the widely distributed myxomycete species Ph. albescens represents a complex of at least 18 cryptic species, and some of these seem to have a limited geographical distribution. In addition, the presence of groups of presumably clonal specimens suggests that sexual and asexual reproduction coexist in natural populations of myxomycetes.     

Phylogenetic relationships and biogeography of Ptilophora (Gelidiales,Rhodophyta) with descriptions of P. aureolusa,P. malagasya,and P. spongiophila from Madagascar

The genus Ptilophora currently includes 16 species occurring mostly in subtidal habitats of the Indo‐Pacific Ocean, but its global diversity and biogeography are poorly understood. We analyzed mitochondrial cox1, plastid rbcL and plastid psbA sequences from specimens collected in southern Madagascar during the 2010 Atimo Vatae expedition and studied their morphologies. Both morphological and molecular data sets demonstrated the presence of five species in southern Madagascar: Ptilophora hildebrandtii, P. pterocladioides, and three new species described here, P. aureolusa, P. malagasya, and P. spongiophila. Ptilophora aureolusa is distinguished by its compound pinnae with uniformly spaced pinnules. Ptilophora malagasya has an indistinct midrib and irregularly spaced pinnules. Ptilophora spongiophila, heavily coated with sponges, has cylindrical to flattened main axes, lateral and surface proliferations, and spatulate tetrasporangial sori. The species of Ptilophora found in Madagascar are endemic, except P. hildebrandtii, which also occurs in eastern Africa. Ptilophora comprises four phylogenetic groups that map to eastern Australia, Japan, western Australia/Southeast Asia/Madagascar/eastern Africa, and Madagascar/eastern Africa/Aegean Sea. Biogeographical analysis revealed that the ancestor of Ptilophora originated in Australia, but most of the species radiated from Madagascar.     

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister‐like glycostyles with hexagonal base and dome‐shaped apex; besides, cell surface bears hair‐like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.     

Ligulate Desmarestia (Desmarestiales,Phaeophyceae) revisited: D. japonica sp. nov. and D. dudresnayi differ from D. ligulata

The phylogeny of ligulate and sulfuric‐acid containing species of Desmarestia, occurring worldwide from polar to temperate regions, was revised using a multigenic and polyphasic approach. Sequence data, gametophyte characteristics, and sporophyte morphology support reducing a total of 16 taxa to four different species. (1) D. herbacea, containing broad‐bladed and highly branched forms, has dioecious gametophytes. The three other species have monoecious gametophytes: (2) D. ligulata which is profusely branched and, except for one subspecies, narrow‐bladed, (3) Japanese ligulate Desmarestia, here described as D. japonica sp. nov., which is morphologically similar to D. ligulata but genetically distant from all other ligulate taxa. This species may have conserved the morphology of original ligulate Desmarestia. (4) D. dudresnayi, including unbranched or little branched broad‐bladed taxa. A figure of the holotype of D. dudresnayi, which was lost for decades, was relocated. The taxonomy is complemented by a comparison of internal transcribed spacer and cytochrome c oxidase subunit I (cox1) as potential barcode loci, with cox1 offering good resolution, reflecting species delimitations within the genus Desmarestia.     

Patterns of diversification in a North American endemic fish,the Blackbanded Darter (Perciformes,Percidae)

The coastal plain of the south‐eastern United States shows multiple biogeographic patterns of plant and animal dispersal; however, few freshwater fish taxa span these biogeographic barriers. Percina nigrofasciata, the Blackbanded Darter (Teleostomi: Percidae), is a small, benthic, freshwater fish species with an extensive range in the south‐eastern United States. Recently, two species have been elevated from within P. nigrofasciata: P. crypta and P. westfalli, but their ranges have not been established. We broadly sampled across the south‐eastern United States, encompassing the range of P. nigrofasciata sensu lato. We reconstruct the phylogeny of Percina using both mitochondrial and nuclear markers. Eighty‐four specimens of Percina nigrofasciata were sampled for the mitochondrial gene cytochrome b (1,119 bp) to form a base phylogeny. The nuclear marker S7‐I1 was subsampled across populations to detect instances of hybridization. Phylogenetic relationships with other members of the genus Percina were assessed through Bayesian inference. Our results suggest that Percina nigrofasciata sensu stricto occurs from the Lake Pontchartrain Basin in Louisiana to the rivers of the Mobile Basin with little genetic structuring throughout its range. Percina westfalli occurs from the Apalachicola River drainages to the Atlantic Slope from the Savannah River to the St. Johns River. We find that P. crypta is not genetically distinct from P. westfalli in the Chattahoochee and Flint Rivers. Possible ancestral hybridization occurred between the P. nigrofasciata and P. westfalli in the panhandle of Florida between Mobile Bay and the Apalachicola River.     

STUDIES ON THE MATING SYSTEMS OF ELEVEN SPECIES OF MYXOMYCETES

Genetic analysis of 20 isolates representing 11 species of Myxomycetes was carried out to determine whether they were heterothallic or homothallic. Each of the single isolates of Badhamia utricularis, Didymium minus, D. nigripes, and Physarum cinereum was found to be heterothallic, the first such reports for all of the species except D. nigripes. All isolates of Badhamia foliicola (1), Didymium squamulosum (1), Physarum compressum (3), P. gyrosum (1), P. pusillum (2), Stemonitis flavogenita (6) and an unidentified species of the order Physarales (2) were homothallic, with B. foliicola, P. compressum, and S. flavogenita being reported for the first time.     

Phylogeny of the Centrohelida Inferred from SSU rRNA, Tubulins, and Actin Genes

Amoeboid protists are major targets of recent molecular phylogeny in connection with reconstruction of global phylogeny of eukaryotes as well as the search for the root of eukaryotes. The Centrohelida are one of the major groups of Heliozoa, classified in the Actinopodida, whose evolutionary position is not well understood. To clarify the relationships between the Centrohelida and other eukaryotes, we sequenced SSU rRNA, α-tubulin, and β-tubulin genes from a centroheliozoan protist, Raphidiophrys contractilis. The SSU rRNA phylogeny showed that the Centrohelida are not closely related to other heliozoan groups, Actinophryida, Desmothoracida, or Taxopodida. Maximum likelihood analyses of the combined phylogeny using a concatenate model for an α- + β-tubulin + actin data set, and a separate model for SSU rRNA, α- and β-tubulin, and actin gene data sets revealed the best tree, in which the Centrohelida have a closer relationship to Rhodophyta than to other major eukaryotic groups. However, both weighted Shimodaira–Hasegawa and approximately unbiased tests for the concatenate protein phylogeny did not reject alternative trees in which Centrohelida were constrained to be sisters to the Amoebozoa. Moreover, alternative trees in which Centrohelida were placed at the node branching before and after Amoebozoa or Viridiplantae were not rejected by the WSH tests. These results narrowed the possibilities for the position of Centrohelida to a sister to the Rhodophyta, to the Amoebozoa, or to an independent branch between the branchings of Amoebozoa and Rhodophyta (or possibly Plantae) at the basal position within the bikonts clade in the eukaryotic tree. [Reviewing Editor: Dr. Martin Kreitman]     

Comparative studies on the diversity of the edible mushroom Pleurotus eryngii: ITS sequence analysis, RAPD fingerprinting, and physiological characteristics

Verification of Pleurotus eryngii strains was assessed using ITS sequence analysis and RAPD fingerprinting. Sequence analysis of the ITS1–5.8S rDNA–ITS2 region of 24 strains of Pleurotus sp., which consisted of 22 strains of P. eryngii and the control strains P. ostreatus and P. ferulae, demonstrated that the DNA regions share mostly 99 % sequence identity, indicating that sequence-based analysis is not applicable for the verification of closely related mushroom strains. To verify the mushroom strains using RAPD, we amplified DNA fragments from the total cellular DNA of 24 mushroom strains with 18 different random primers, yielding 538 distinct DNA fragments ranging from 200–4000 bp. Analysis of the DNA fragment pattern showed that the 22 P. eryngii strains were clearly distinguished from the control strains P. ostreatus and P. ferulae, and could be categorized into five subgroups. Subsequent physiological studies on the development of fruiting bodies demonstrated the close correlation of the RAPD-based grouping with the phenotypical characteristics of mushroom fruiting bodies.     

Species‐delimitation and phylogenetic analyses of some cosmopolitan species of Hypnea (Rhodophyta) reveal synonyms and misapplied names to H. cervicornis,including a new species from Brazil

Hypnea has an intricate nomenclatural history due to a wide pantropical distribution and considerable morphological variation. Recent molecular studies have provided further clarification on the systematics of the genus; however, species of uncertain affinities remain due to flawed taxonomic identification. Detailed analyses coupled with literature review indicated a strong relationship among H. aspera, H. cervicornis, H. flexicaulis, and H. tenuis, suggesting a need for further taxonomic studies. Here, we analyzed sequences from two molecular markers (COI‐5P and rbcL) and performed several DNA‐based delimitation methods (mBGD, ABGD, SPN, PTP and GMYC). These molecular approaches were contrasted with morphological and phylogenetic evidence from type specimens and/or topotype collections of related species under a conservative approach. Our results demonstrate that H. aspera and H. flexicaulis represent heterotypic synonyms of H. cervicornis and indicate the existence of a misidentified Hypnea species, widely distributed on the Brazilian coast, described here as a new species: H. brasiliensis. Finally, inconsistencies observed among our results based on six different species delimitation methods evidence the need for adequate sampling and marker choice for different methods.     

Glomerulosomes: morphologically distinct nuclear organelles of unknown nature

The nucleus of some representatives of the genus Pelomyxa (Amoebozoa, Archamoebae, Pelobiontida) contains specific bodies (membrane-less organelles). They may be either embedded in the nucleolar mass or detached from the nucleolus. We termed these nuclear bodies the glomerulosomes for their characteristic ultrastructural appearance. The glomerulosomes are distinct nuclear bodies, about 1 μm in diameter. The morphological and diagnostic unit of a glomerulosome is an electron-dense thread/string, about 30–40 nm in thickness. These threads are not direct continuation of the nucleolar material. The threads create the unique geometric appearance of the glomerulosome by being organized into precisely parallel rows/cords. Each cord of the threads can curve at different angles within the glomerulosome body, but the threads themselves are not coiled. Nowadays, the glomerulosomes have been discovered in P. palustris, P. stagnalis, P. paradoxa, and Pelomyxa sp. Despite the unique appearance of glomerulosomes, their existence may be a more common phenomenon in eukaryotic cells than just a specific feature of the nucleus of elected pelomyxes.

     

DNA Sequencing of Type Material Reveals Pneophyllum marlothii comb. nov. from South Africa and P. discoideum comb. nov. (Chamberlainoideae,Corallinales, Rhodophyta) from Argentina

A partial rbcL sequence from the type material of Spongites discoideus from southern Argentina showed that it was distinct from rbcL sequences of South African specimens to which that name had been applied based on morpho-anatomy. A partial rbcL sequence from an original syntype specimen, herein designated the lectotype, of Lithophyllum marlothii, type locality Camps Bay, Western Cape Province, South Africa, was identical to rbcL sequences of South African field-collected specimens assigned to S. discoideus. Based on phylogenetic analyses of rbcL and/or psbA sequences, both of these species belong in Pneophyllum and are transferred there as P. discoideum comb. nov. and P. marlothii comb. nov. The two species exhibit a distinct type of development where thick, secondary, monomerous disks are produced from thin, primary, dimerous crusts. Whether this type of development represents an example of convergent evolution or is characteristic of a clade of species within Pneophyllum remains to be resolved.     

Variation in the SSUrDNA of the Genus Protostelium Leads to a New Phylogenetic Understanding of the Genus and of the Species Concept for Protostelium mycophaga (Protosteliida,Amoebozoa)

Members of the genus Protostelium (including P. mycophaga, P. nocturnum, and P. okumukumu) are protosteloid amoebae commonly found in terrestrial habitats on dead plant matter. They, along with the closely allied nominal genus Planoprotostelium, containing the single species Pl. aurantium, all have an amoeboid trophic stage with acutely pointed subpseudopodia and orange lipid droplets in the granuloplasm. These amoebae form stalked fruiting bodies topped with a single, usually deciduous spore. The species are identified based on their fruiting body morphologies except for Pl. aurantium which looks similar to P. mycophaga in fruiting morphology, but has amoebae that can make flagella in liquid medium. We built phylogenetic trees using nuclear small subunit ribosomal DNA sequences of 35 isolates from the genera Protostelium and Planoprotostelium and found that (1) the nonflagellated P. nocturnum and P. okumukumu branch basally in the genus Protostelium, (2) the flagellate, Pl. aurantium falls within the genus Protostelium in a monophyletic clade with the nominal variety, P. mycophaga var. crassipes, (3) the cultures initially identified as Protostelium mycophaga can be divided into at least three morphologically recognizable taxa, P. aurantium n. comb., P. apiculatum n. sp., and P. m. rodmani n. subsp., as well as a paraphyletic assemblage that includes the remainder of the P. mycophaga morphotype. These findings have implications for understanding the ecology, evolution, and diversity of these amoeboid organisms and for using these amoebae as models for other amoeboid groups.     

Fatty acid profiles of polar and non-polar lipids of Pleurotus ostreatus and P. cornucopiae var. ‘citrino-pileatus’ grown at different temperatures

The application of fatty acid (FA) composition data has now extended to studies of physiology, chemotaxonomy, and intrageneric differentiation, as well as to studies of human nutrition. Environmental factors such as nutritional components, oxygen, and temperature are known to affect lipid content and composition in living organisms, including fungi. In the present study, the polar and non-polar lipid content of Pleurotus ostreatus and P. cornucopiae var. citrino-pileatus fruiting bodies produced at temperatures ranging from 12–27 °C and from 17–27 °C, respectively, were analysed to evaluate the effect of temperature on lipid composition in these mushrooms. Results showed that lowering the growth temperature below 17 °C generally provided an expected increase in FA unsaturation in polar and non-polar lipids of P. ostreatus. Although raising the temperature above 17 °C did not show any clear-cut tendency in FA unsaturation, it did reveal that growth temperature had a differential effect on the FA profiles in fruiting bodies of P. ostreatus and P. cornucopiae. This study suggests that care should be taken when using FA content and unsaturation data for physiological, chemotaxonomic, and intrageneric differentiation studies, and that it may be possible to manipulate lipid unsaturation in Pleurotus spp. through modified growth temperatures.