Laboratory culture and bioactive natural products of myxomycetes.

Myxomycetes, one of the lowest classes of eukaryote (true slime molds), are an unusual group of primitive organisms. Their life cycle consists of two stages, namely the free-living plasmodium and the fruiting body with unique structures and colors. The chemical studies on the secondary metabolites of the myxomycetes are limited due to a lack of understanding of their laboratory cultivation. In this review, 93 natural products from myxomycetes, including their chemical structures and bioactivities were described. We also provided a conceptual overview over five culture methods of myxomycetes, including moist chamber culture, feeding culture, pure culture, liquid culture and hanging drop culture.

Nivicolous Trichiales from the austral Andes: unexpected diversity including two new species.

Nivicolous myxomycetes are a group of amoebozoan protists dependent on long-lasting snow cover worldwide. Recent fine-scale analysis of species diversity from the austral Andes revealed high intraspecific variability of most taxa, suggesting independent evolutionary processes and significant differences in species compositions between the Northern (NH) and Southern (SH) Hemispheres. The present study is the second part of this analysis based on representatives of Trichiales. A total of 173 South American collections were studied based on morphological and molecular data, and 15 taxa have been identified. Two of them, Hemitrichia crassifila and Perichaena patagonica, are proposed as new species confirmed by a phylogeny of Trichiales. However, their affinity to the genera in which they are proposed are not confirmed due to polyphyletic character of all genera of Trichiales. Four species, Dianema subretisporum, Trichia contorta var. karstenii, T. nivicola, and T. sordida, are reported for the first time from the Southern Hemisphere. One species, T. alpina, is new for Argentina. Additionally, we provide the first record of Perichaena megaspora from Chile. Specimen frequency and species diversity of Trichiales found at nivicolous localities in the austral Andes are unexpectedly high, exceeding those of Stemonitidales, the most numerous group in the Northern Hemisphere, where Trichiales play a marginal role. By contrast, Trichiales appear the main component of nivicolous assemblages in the Andes. Results of the present work, together with the earlier analysis of Stemonitidales, indicate that the Andes constitute an exceptionally important evolutionary hot spot for nivicolous myxomycetes characterized by an outstanding species diversity.

Loss of the Polyketide Synthase StlB Results in Stalk Cell Overproduction in Polysphondylium violaceum.

Major phenotypic innovations in social amoeba evolution occurred at the transition between the Polysphondylia and group 4 Dictyostelia, which comprise the model organism Dictyostelium discoideum, such as the formation of a new structure, the basal disk. Basal disk differentiation and robust stalk formation require the morphogen DIF-1, synthesized by the polyketide synthase StlB, the des-methyl-DIF-1 methyltransferase DmtA, and the chlorinase ChlA, which are conserved throughout Dictyostelia. To understand how the basal disk and other innovations evolved in group 4, we sequenced and annotated the Polysphondylium violaceum (Pvio) genome, performed cell type-specific transcriptomics to identify cell-type marker genes, and developed transformation and gene knock-out procedures for Pvio. We used the novel methods to delete the Pvio stlB gene. The Pvio stlB- mutants formed misshapen curly sorogens with thick and irregular stalks. As fruiting body formation continued, the upper stalks became more regular, but structures contained 40% less spores. The stlB- sorogens overexpressed a stalk gene and underexpressed a (pre)spore gene. Normal fruiting body formation and sporulation were restored in Pvio stlB- by including DIF-1 in the supporting agar. These data indicate that, although conserved, stlB and its product(s) acquired both a novel role in the group 4 Dictyostelia and a role opposite to that in its sister group.

The Response of Litter-Associated Myxomycetes to Long-Term Nutrient Addition in a Lowland Tropical Forest.

Myxomycetes (plasmodial slime molds) are abundant protist predators that feed on bacteria and other microorganisms, thereby playing important roles in terrestrial nutrient cycling. Despite their significance, little is known about myxomycete communities and the extent to which they are affected by nutrient availability. We studied the influence of long-term addition of N, P, and K on the myxomycete community in a lowland forest in the Republic of Panama. In a previous study, microbial biomass increased with P but not N or K addition at this site. We hypothesized that myxomycetes would increase in abundance in response to P but that they would not respond to the sole addition of N or K. Moist chamber cultures of leaf litter and small woody debris were used to quantify myxomycete abundance. We generated the largest myxomycete dataset (3,381 records) for any single locality in the tropics comprised by 91 morphospecies. In line with our hypothesis, myxomycete abundance increased in response to P addition but did not respond to N or K. Community composition was unaffected by nutrient treatments. This work represents one of very few large-scale and long-term field studies to include a heterotrophic protist highlighting the feasibility and value in doing so.

A Method for Growing Bio-memristors from Slime Mold.

Our research is aimed at gaining a better understanding of the electronic properties of organisms in order to engineer novel bioelectronic systems and computing architectures based on biology. This specific paper focuses on harnessing the unicellular slime mold Physarum polycephalum to develop bio-memristors (or biological memristors) and bio-computing devices. The memristor is a resistor that possesses memory. It is the 4th fundamental passive circuit element (the other three are the resistor, the capacitor, and the inductor), which is paving the way for the design of new kinds of computing systems; e.g., computers that might relinquish the distinction between storage and a central processing unit. When applied with an AC voltage, the current vs. voltage characteristic of a memristor is a pinched hysteresis loop. It has been shown that P. polycephalum produces pinched hysteresis loops under AC voltages and displays adaptive behavior that is comparable with the functioning of a memristor. This paper presents the method that we developed for implementing bio-memristors with P. polycephalum and introduces the development of a receptacle to culture the organism, which facilitates its deployment as an electronic circuit component. Our method has proven to decrease growth time, increase component lifespan, and standardize electrical observations.

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.

What an Intron May Tell: Several Sexual Biospecies Coexist in Meriderma spp. (Myxomycetes).

Specimens of the snowbank myxomycete Meriderma atrosporum agg. from five European mountain ranges were sequenced for parts of the nuclear small subunit ribosomal RNA gene (SSU) and the protein elongation factor 1 alpha gene (EF1A). A phylogeny of the EF1A gene, including a very variable spliceosomal intron, resulted in seven phylogroups, and this topology was confirmed by SSU sequences. Two thirds of all specimens were heterozygous for the EF1A gene, and the two haplotypes of these specimens occurred always in the same phylogroup. Except for two cases in closely related phylogroups all ribotypes were as well limited to one phylogroup. This pattern is consistent with the assumption of reproductively isolated sexual biospecies. Numbers of EF1A-haplotypes shared between mountain ranges correlate with geographical distance, suggesting relative isolation but occasional long-distance dispersal by spores. Most subpopulations (divided by putative biospecies and mountain ranges) were in Hardy-Weinberg equilibrium. A simulation assuming panmixis within but not in between subpopulations suggested that similar numbers of shared genotypes can be created by chance through sexual reproduction alone. Our results support the biospecies concept, derived from experiments with cultivable members of the Physarales. We discuss the results on the background of possible reproductive options in myxomycetes.

On chirality of slime mould.

Left-right patterning and lateralised behaviour is an ubiquitous aspect of plants and animals. The mechanisms linking cellular chirality to the large-scale asymmetry of multicellular structures are incompletely understood, and it has been suggested that the chirality of living cells is hardwired in their cytoskeleton. We examined the question of biased asymmetry in a unique organism: the slime mould Physarum polycephalum, which is unicellular yet possesses macroscopic, complex structure and behaviour. In laboratory experiment using a T-shape, we found that Physarum turns right in more than 74% of trials. The results are in agreement with previously published studies on asymmetric movement of muscle cells, neutrophils, liver cells and growing neural filaments, and for the first time reveal the presence of consistently-biased laterality in the fungi kingdom. Exact mechanisms of the slime mould's direction preference remain unknown.

Nivicolous Stemonitales from the Austral Andes: analysis of morphological variability, distribution and phenology as a first step toward testing the large-scale coherence of species and biogeographical properties.

Nivicolous myxomycetes occur at the edge of spring-melting snow in mountainous areas. They are mostly considered cosmopolitan species morphologically and ecologically uniform across their entire distribution ranges. Thus, long-distance dispersal has been suggested to be the main mechanism shaping their ranges and geographical variability patterns. To test this hypothesis we conducted the first detailed analysis of morphological variability, occurrence frequency and phenology of nivicolous myxomycetes collected in the hitherto unexplored Austral Andes of South America (southern hemisphere = SH) in the comparative context of data from the northern hemisphere (NH). We used Stemonitales, the most representative and numerous taxonomic order in nivicolous myxomycetes, as a model. A total of 131 South American collections represented 13 species or morphotypes. One of them, Lamproderma andinum, is new to science and described here. Several others, L. aeneum, L. album, L. pulveratum, "Meriderma aff. aggregatum ad. int.", M. carestiae and "M. spinulosporum ad. int.", were previously unknown from the SH. Lamproderma ovoideum is reported for the first time from South America and Collaria nigricapillitia is new for Argentina. The fine-scale morphological analysis of all species from the study area and reference NH material demonstrated a high intraspecific variability in most of them. This suggests isolation and independent evolutionary processes among remote populations. On the other hand, the uniform morphology of a few species indicates that long-distance dispersal is also an effective mechanism, although not as universal as usually assumed, in some nivicolous myxomycetes. Analysis of nivicolous species assemblages also showed significant differences among major geographic regions in that the Stemonitales were significantly less common in the SH than in the NH. Furthermore, the occurrence of nivicolous species in summer and autumn, out of the typical phenological season, is recognized as a possible distinctive phenomenon for the SH populations.

Didymium xerophilum, a new myxomycete from the tropical Andes.

A new species of Didymium (Myxomycetes), D. xerophilum, is described, and some details of its life cycle are provided. The new species was collected during studies of arid areas of Argentina and Peru. It can be distinguished by the persistent funnel-shaped invagination of the peridium, the top of which appears as a deep umbilicus in closed sporothecae, and the calcareous hypothallus shared among several sporocarps. This combination of characters, with a circumscissile dehiscence of the sporotheca and a cream stalk packed with rhombic lime crystals, is unknown in other described species. Morphology was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included here. Phylogenetic analysis with 18S rDNA sequences of different species of Didymium supports the distinct identity of this new species. Some collections of this myxomycete were made at up to 4600 m, an altitude almost unknown for this group of microorganisms.

Evaluating the potential use of myxomycetes as a source of lipids for biodiesel production.

The myxomycetes are a group of primitive phagotrophic eukaryotes characterized by a distinctive plasmodial stage that is well known for its rapid growth rate. In the present study, biomass and lipid production of several different species of myxomycetes were investigated. Physarum polycephalum was found to produce the highest amounts of both dry biomass (1.30g), and lipid (0.143g) per 20mL medium (equal to 65.0g biomass and 7.15g lipid per one liter of medium). Analysis of P. polycephalum lipids by thin layer chromatography (TLC) and fatty acid methyl esters (FAMES) by gas chromatography-mass spectrometry (GC-MS) techniques showed that the major lipid type is triglyceride (95.5%), followed by phospholipids (2.6%); diglyceride (0.92%) and monoglyceride (0.92%). Myxomycete lipids consist of three dominant fatty acids: oleic (20%), linoleic (33%), and palmitoleic (17%). These results suggest that P. polycephalum has considerable potential as a source of lipids for biodiesel production.

Comparison of wood-inhabiting myxomycetes in subalpine and montane coniferous forests in the Yatsugatake Mountains of Central Japan.

To demonstrate altitudinal gradients (and resulting temperatures) that affect myxomycete biodiversity and species composition, we statistically compared myxomycete assemblages between a subalpine coniferous forest and a montane pine forest within the region of the Yatsugatake Mountains, Nagano Prefecture, Central Japan. In summer and autumn field surveys during 2003-2010, 53 myxomycete taxa (with varieties treated as species) were observed from 639 records of fruiting bodies in the subalpine forest and 32 taxa were detected from 613 records in the montane forest. There were 20 species in common between the assemblages and the percentage similarity index was 0.400. Myxomycete biodiversity was higher in the subalpine than in the montane forest. Nine myxomycete species were statistically frequent occurrences in the subalpine forest and appeared in autumn: Lamproderma columbinum, Cribraria macrocarpa, Trichia botrytis, Physarum newtonii, Diderma ochraceum, Enteridium splendens, Elaeomyxa cerifera, Trichia verrucosa, and Colloderma oculatum. Five species were restricted to appear in the subalpine forest: Cribraria purpurea, Cribraria rufa, Cribraria ferruginea, Cribraria piriformis, and Lepidoderma tigrinum. Dead wood in the subalpine forest provided a breeding habitat for specific myxomycetes that inhabit cold areas; that is those areas having geographical features of decreasing temperature and increasing elevation, such as the temperate area of Central Japan.

Spore to spore culture of Didymium operculatum, a new Myxomycete from the Atacama Desert of Chile.

A new species of Didymium (Myxomycetes), D. operculatum, is described in this paper, and details of its life cycle are provided. The new species was recorded during studies of the Atacama Desert in Chile. It has been collected directly in the field and isolated in moist chamber cultures prepared with material from an endemic cactus. The distinguishing characters of this species are its dehiscence by means of an apical operculum combined with a whitish calcareous stalk and the banded reticulate ornamentation of the spores. The morphology of this new myxomycete was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included in this paper. Some comments are made on the patterns of distribution of Didymium species in arid lands and adaptive characters enabling this genus to colonize such extreme environments. It is proposed that a longer cycle and the ability to resort to resistant forms many times during their development reflect the response of these myxomycetes to the largely unfavorable conditions of their environment.

Two new species of nivicolous Lamproderma (myxomycetes) from the mountains of Europe and America.

As a result of the revision of European and American collections of genus Lamproderma two new nivicolous myxomycete species, Lamproderma argenteobrunneum and L. kowalskii, are described. The new species are characterized by the silvery-brown sporothecae, the areolate peridium and the ferruginous-brown spores in mass. They differ from one another mainly in spore size and ornamentation, the form of the capillitium and the stalk length and ratio of the stalk length to the total height of the sporocarp. The morphology of the new species was examined with light microscopy and scanning electron microscopy (SEM), and micrographs of relevant details are included. Lamproderma argenteobrunneum also was obtained in moist chamber culture, and the mature sporocarps displayed all features typical of field-collected samples. The known geographical distribution of L. argenteobrunneum includes the main ranges of the European alpine system (Alps, Carpathians, Pyrenees) as well as those of North America, while L. kowalskii has been recorded so far from several sites in California in the United States.

Description and life cycle of a new Didymium (Myxomycetes) from arid areas of Argentina and Chile.

A new species of Didymium (Myxomycetes), D. infundibuliforme, is described herein, and details are provided on its life cycle as observed in spore to spore culture on agar. The new species was recorded during intensive studies of areas of the Monte Desert in Argentina and the Atacama Desert in Chile. It has been collected directly in the field in both countries on several occasions over 4 y and isolated in moist chamber cultures prepared with material from native plant species. The characters that make this species unique in the genus are its funnel-shape sporocarps with white stalks, the apical circumscissile dehiscence of the sporotheca that causes the base to resemble a calyculus and the ornamentation on the spores. The morphology of specimens of this new myxomycete was examined with scanning electron microscopy and light microscopy, and micrographs of relevant details are included in this paper.

First records and microhabitat assessment of protostelids in the Aberdare Region, Central Kenya.

A rapid assessment survey on the occurrence and distribution of protosteloid amoebae was carried out in central Kenya. Samples of dead plant materials were collected from 46 study sites (each 20 x 20 m) situated along an elevation gradient (1,785-3,396 m) that encompassed five major land use/cover types. Twenty-four species and subspecific taxa were recovered and included 23 protostelids and one minute myxomycete, often included in surveys for protostelids. All of these were the first records for Kenya, and six were new for Africa. Numbers of taxa were highest in ground litter and aerial litter microhabitats (20 taxa each) and lowest on aerial bark (10) and ground bark (7). Relative species abundance was greatest in aerial litter, moderate in ground litter, and low on aerial and ground bark microhabitats. The most frequently occurring species on ground litter were Schizoplasmodiopsis pseudoendospora, Schizoplasmodiopsis amoeboidea, and Protostelium mycophaga var. mycophaga, whereas the most common species on aerial litter were P. mycophaga var. mycophaga and Soliformovum irregularis. Species richness and abundance decreased with increasing elevation.

Distribution and occurrence of myxomycetes on agricultural ground litter and forest floor litter in Thailand.

The ecological distribution and seasonal patterns of occurrence of myxomycetes associated with the microhabitats represented by agricultural ground litter and forest floor litter were investigated in six study sites in northern Thailand during Oct 2004-Oct 2005. Both specimens that developed under natural conditions in the field and specimens obtained in moist chamber cultures were considered. Agricultural study sites were a banana plantation, a mango orchard and a sweet corn farm, whereas forest study sites consisted of a lowland forest, a midelevation forest and a highland forest. Seventy species of myxomycetes representing 20 genera were collected, with 34 species recorded during the dry season and 58 species recorded during the rainy season. Distinct differences were apparent for numbers of positive moist chambers, numbers of species recorded from moist chambers, numbers of species recorded as field collections and numbers of field collections when data obtained for the two seasons were compared. In the dry season only three species were collected in the field, but the percentage of positive moist chamber cultures (129/180 or 72%) and number of species recorded (33) from these cultures were high. In contrast, during the rainy season, 52 species of myxomycetes were collected in the field but only 15 species were recorded from the much lower percentage (75/180 or 42%) of positive moist chambers cultures.

Influence of bark pH on the occurrence and distribution of tree canopy myxomycete species.

This study compares the occurrence and distribution of myxomycete species in the canopy of living trees and neighboring grapevines. Corticolous myxomycetes of three temperate forests in southeastern USA were studied on six tree species (30 trees) and grapevines (30 vines) to determine distribution and occurrence of myxomycete species relating to geographic location, host species, and bark pH. The double-rope climbing technique was used to access the canopy and sample bark up to 16.5 m. Bark samples were examined in 580 moist chamber cultures and 44 myxomycete species were identified representing 21 genera, averaging 3.0 +/- 2.1 species per sample site. Jaccard's coefficient determined community similarity between five individuals of six tree species, Acer saccharum, Fraxinus americana, Liquidambar styraciflua, Liriodendron tulipifera, Platanus occidentalis and Tsuga canadensis, and neighboring grapevines, Vitis aestivalis and V. vulpina. Vertical variation in species richness was significantly different only for Platanus occidentalis and might be attributable to flaking of bark with increasing height in the canopy. Tsuga canadensis and neighboring grapevines had greatest community similarity. Cribraria violacea was observed on all tree and grapevine species except T. canadensis and neighboring grapevines. Occurrence and species assemblages of myxomycetes were associated with bark pH, not geographic location. Bark of V. aestivalis (pH 4.5) was more acidic than neighboring T. canadensis (pH 4.1), compared to grapevines of the same species neighboring other tree species. Results indicated that most species are not regionally restricted, and although some myxomycetes are associated with a certain pH range, others develop on any substratum. Future research protocols for corticolous myxomycetes should emphasize sampling adequate amounts of substrata in a local region from different host species that have a wide range of bark pH, ensuring a representative sample of species for an entire region.

A habitat colonisation model for spore-dispersed organisms: does it work with eumycetozoans?

Spore productivities and establishment probabilities of eumycetozoans were estimated and compared with quantitative data obtained from field surveys, using series of cultures of a given substrate. Spore numbers per spore case were found to increase from one to four in protostelids to up to 10(5)-10(6) in myxomycetes, whereas average spore size decreased slightly from 14.8 microm for protostelids to 10.3 microm in myxomycetes. Spore numbers of fructifications calculated from dimensions of spores and fruit bodies were in good agreement with direct counts carried out for six species of myxomycetes. A colonisation model is presented that estimates frequencies (as a percent of successfully colonized habitat islands), which is independent of a given density of spore rain and the sexual system of the species being considered. Whereas asexual species need a minimum spore rain of ca 0.7 spores per habitat island to reach a frequency of 50%, this figure is at least 2.4-fold higher for sexual species, depending from the incompatibility system assumed. Data from cultures indicate that the maximum potential spore rain is usually three orders of magnitude higher than the minimum figure required to create the observed frequencies. Eumycetozoans seem to follow the evolutionary trends predicted by the model. Species with sexual reproductive systems produce often more spores than asexual ones; many morphospecies have sexual and asexual strains; and back-conversion from sexual to asexual reproduction occurs occasionally.

Evolution of dark-spored Myxomycetes (slime-molds): molecules versus morphology.

The Myxomycetes are a major component of soil amoebae, displaying a complex life cycle that terminates in the formation of often macroscopic fruiting bodies. The classification of Myxomycetes is controversial and strongly depends on the weight given by different authors to morphological and developmental characters. We used a molecular approach to establish the phylogenetic relationships in the dark-spored orders Stemonitales and Physarales. Twenty-five small subunit ribosomal RNA gene sequences were obtained, with focus on two Stemonitales genera, Lamproderma and Comatricha. Unexpectedly, our results show that Stemonitales are paraphyletic with Physarales arising from within a Lamproderma clade. The genus Lamproderma itself is polyphyletic and can be divided into two distinct clades. Additionally, we found that Comatricha nigricapillitia comprises two cryptic species, both related to Enerthenema. Our study allows the reappraisal of morphological and developmental characters in the light of molecular data and sets foundations for a new classification of Myxomycetes.