Thin film structural color is widespread in slime molds (Myxomycetes, Amoebozoa).

Brilliant colors in nature arise from the interference of light with periodic nanostructures resulting in structural color. While such biological photonic structures have long attracted interest in insects and plants, they are little known in other groups of organisms. Unexpected in the kingdom of Amoebozoa, which assembles unicellular organisms, structural colors were observed in myxomycetes, an evolutionary group of amoebae forming macroscopic, fungal-like structures. Previous work related the sparkling appearance of Diachea leucopodia to thin film interference. Using optical and ultrastructural characterization, we here investigated the occurrence of structural color across 22 species representing two major evolutionary clades of myxomycetes including 14 genera. All investigated species showed thin film interference at the peridium, producing colors with hues distributed throughout the visible range that were altered by pigmentary absorption. A white reflective layer of densely packed calcium-rich shells is observed in a compound peridium in Metatrichia vesparium, whose formation and function are still unknown. These results raise interesting questions on the biological relevance of thin film structural colors in myxomycetes, suggesting they may be a by-product of their reproductive cycle.

Importance of type specimen study for understanding genus boundaries-taxonomic clarifications in Lepidoderma based on integrative taxonomy approach leading to resurrection of the old genus Polyschismium.

Type specimens of four species of Lepidoderma (Myxomycetes, Amoebozoa)-L. crassipes, L. neoperforatum, L. perforatum, and L. stipitatum-have been studied using an integrative approach including application of traditional taxonomy methods, i.e., morphological study under stereoscopic and compound microscopes, detailed analysis of micromorphological characters using scanning electron microscopy, and molecular analysis by way of Sanger sequencing of molecular markers (nuc 18S rDNA and elongation factor 1-alpha gene, EF1A). Results of the study revealed that L. crassipes is conspecific with L. tigrinum, L. stipitatum is a malformed specimen of Diderma floriforme, whereas L. perforatum and L. neoperforatum represent two well-defined morphologically and genetically separate species. Phylogeny of Physarales shows the polyphyletic character of the genus Lepidoderma. The type species of Lepidoderma clusters together with Diderma, whereas other representatives of this genus form a monophyletic, well-supported clade. The species from this clade are proposed to belong to the genus Polyschismium described by A. Corda in 1842 that is resurrected and emended here. Nine species of Lepidoderma are transferred to Polyschismium. A new key to Didymiaceae including Polyschismium is provided.

Didymium pseudonivicola: A new myxomycete from the austral Andes emerges from broad-scale morphological and molecular analyses of D. nivicola collections.

A new nivicolous myxomycete is described as a result of a comprehensive study of Didymium nivicola collections from the entire range of its occurrence. Statistical analysis of 12 morphological characters, phylogenetic analyses of nuc 18S rDNA and elongation factor 1-alpha gene (EF1A), and a delimitation method (automatic barcode gap diversity) have been applied to corroborate the identity of the new species. A preliminary morphological analysis of D. nivicola revealed high variability of South American populations where four types of spore ornamentation were noted. However, results of molecular study and statistical analysis of morphological characters did not support recognition of these four forms but the distinction of two morphotypes. Consequently, two species have been recognized: D. nivicola and the newly proposed D. pseudonivicola. The new species can be distinguished from D. nivicola by distinctly larger and mostly plasmodiocarpic sporophores, which are scattered to gregarious, paler spores, and by the paler, more delicate and more elastic capillitium. Spore ornamentation of D. pseudonivicola is uniform and can be described as distinctly spiny (pilate under scanning electron microscope [SEM]), whereas those of D. nivicola is more variable, where spines (pilae under SEM) are delicate, distinct, or conspicuous. Additionally, whereas D. nivicola is a species distributed worldwide, D. pseudonivicola occurs only in the austral Andes of Argentina and Chile.

Range-wide Phylogeography of a Nivicolous Protist Didymium nivicola Meyl. (Myxomycetes, Amoebozoa): Striking Contrasts Between the Northern and the Southern Hemisphere.

Soil protists play a crucial role in terrestrial ecosystems and often show immense taxonomic diversity. However, for many groups, distribution patterns remain largely unknown. We investigated range-wide intraspecific diversity of a specialized airborne protist (Didymium nivicola Meyl.) that occupies a narrow ecological niche associated with long-lasting snow cover. We sampled 122 collections covering all areas where the species was recorded worldwide. We obtained 105 and 41 sequences of small ribosomal subunit rDNA (SSU) and elongation factor 1-alpha (EF1A), respectively. While the species is very diverse in the austral Andes, Southern Hemisphere (SH; 17 SSU ribotypes and 12 EF1A genotypes identified), its populations are genetically uniform across three continents of the Northern Hemisphere (NH; single ribotype, single genotype). Our results indicate the austral Andes as a possible diversification centre for D. nivicola where populations seem to reproduce sexually. Two main parts of the range display highly contrasting genetic patterns, thus biogeographical history and dynamics. Current distribution of D. nivicola in the NH is likely a result of a dispersal event from the SH and subsequent long-distance dispersal (LDD) that might be associated with a shift to asexual mode of reproduction.

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.

New protocol for successful isolation and amplification of DNA from exiguous fractions of specimens: a tool to overcome the basic obstacle in molecular analyses of myxomycetes.

Herbarium collections provide an essential basis for a wide array of biological research and, with development of DNA-based methods, they have become an invaluable material for genetic analyses. Yet, the use of such material is hindered by technical limitations related to DNA degradation and to quantity of biological material. The latter is inherent for some biological groups, as best exemplified by myxomycetes which form minute sporophores. It is estimated that ca. two-thirds of myxomycete taxa are represented by extremely scanty material. As DNA isolation methods applied so far in myxomycete studies require destructive sampling of many sporophores, a large part of described diversity of the group remains unavailable for phylogenetic studies or barcoding. Here, we tested several procedures of DNA isolation and amplification to seek for an efficient and possibly non-destructive method of sampling. Tests were based on herbarium specimens of 19 species representing different taxonomic orders. We assayed several variants of isolation based on silica gel membrane columns, and a newly designed procedure using highly reduced amount of biological material (small portion of spores), based on fine disruption of spores and direct PCR. While the most frequently used column-based method led to PCR success in 89.5% of samples when a large amount of material was used, its performance dropped to 52% when based on single sporophores. Single sporophores provided amplicons in 89.5% of samples when using a kit dedicated to low-amount DNA samples. Our new procedure appeared the most effective (94.7%) while it used only a small fraction of spores, being nearly non-destructive; it was also the most cost-effective. We thus demonstrate that combination of adequate handling of spore micro-disruption coupled with application of direct PCR can be an efficient way to circumvent technical limitations for genetic studies in myxomycetes and thus can substantially improve taxon sampling for phylogeny and barcoding. Additionally, this approach gives a unique possibility to apply both molecular and morphological assays to the same structure (sporophore), which then can be further stored as documentation.

Is Myxomycetes (Amoebozoa) a Truly Ambiregnal Group? A Major Issue in Protist Nomenclature.

Myxomycetes is one of the largest groups of protists belonging to Amoebozoa, with ca 1,000 species recognised and more than 4,000 names in use. Historically, myxomycetes were considered fungi or protozoans which, however, fell under the provisions of the former International Code of Botanical Nomenclature (ICBN), currently the International Code of Nomenclature for algae, fungi, and plants (ICN). Attempts to apply the International Code of Zoological Nomenclature (ICZN) to myxomycetes were rare and inconsistent; thus, we argue that Myxomycetes is not a truly ambiregnal group (i.e. one falling under both Codes). Recently, nomenclatural novelties within Myxomycetes have been proposed using ICZN rules, and the application of zoological orthography to myxomycete higher-level taxa in the recent amoebozoan phylogenies is increasingly common. We summarise the consequences of application of either ICN or ICZN to Myxomycetes. In our opinion, nomenclatural stability within Myxomycetes is best served by strict application of ICN. Either treating myxomycetes as falling under ICZN or considering them an ambiregnal group would cause serious nomenclatural instability, mainly owing to the incompatibility of the two Codes as to the date of the starting point of nomenclature and to the appearance of numerous homonyms.

Molecular inference, multivariate morphometrics and ecological assessment are applied in concert to delimit species in the Russula clavipes complex.

Species of Russula subsect. Xerampelinae are notoriously difficult to identify and name and have not been subject to molecular study. A group of species, referred to here as the R. clavipes complex, growing in association with Salix, Betula and Populus as well as coniferous tree species from temperate to arctic and alpine habitats, were examined. Analyses of the nuc rDNA internal transcribed spacer (ITS) region and a numerical analysis of morphological characters were used. The R. clavipes complex is a monophyletic group within Russula subsect. Xerampelinae, according to molecular results. The complex includes three species: R. nuoljae is a phylogenetically and morphologically well-supported species while the other two, R. clavipes and R. pascua, are similar based on ITS data and morphology but separate based on their ecology. Russula pseudoolivascens is conspecific with R. clavipes Several combinations of characters traditionally used in the taxonomy of R. subsect. Xerampelinae are inappropriate for species delimitation in this group and the adequacy of the ITS for species identification in this group is discussed. Detailed microscopic observations on the type collection of R. nuoljae are presented and illustrated, along with a key to the European members of R. subsect. Xerampelinae.

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.

The genus Hebeloma in the alpine belt of the Carpathians including two new species.

Between 2002 and 2012 regular visits to the Carpathians were made and a number of Hebeloma spp. were collected from the alpine area. In total 44 collections were made that represent 11 species, two of which, Hebeloma grandisporum and H. oreophilum, are described here as new. Of the 11 species, four (H. alpinum, H. marginatulum and the two species described as new) are known only from alpine or Arctic habitats. Hebeloma dunense and H. mesophaeum are commonly found in, but not restricted to, alpine habitats. The other five species (H. aanenii, H. laterinum, H. naviculosporum, H. vaccinum, H. velutipes) are usually found in lowland or boreal habitats. Hebeloma naviculosporum is reported for the first time from the alpine zone and H. alpinum for the first time as growing with Helianthemum. All but two species (H. alpinum, H. marginatulum) are reported for the first time from the Carpathian alpine zone. In this paper we discuss the habitat, the 11 recorded species and give detailed descriptions of the two new species, both morphologically and molecularly. A key for Hebeloma species from sect. Hebeloma occurring in Arctic-alpine habitats is provided.

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.

Perichaena megaspora, a new nivicolous species of myxomycete from the Andes.

A new nivicolous species of Perichaena is described from the Andes in Argentina. The most conspicuous characteristics of Perichaena megaspora are the large spores and their ornamentation in the form of flattened warts. The 16-21 µm diam spores make the new species unique in the genus in which all other species have spores rarely reaching 15 µm diam. Twenty-two collections were found in the field during two consecutive years at 10 localities in Mendoza province Argentina, including one collection isolated from a moist chamber culture of ground litter. The new species was examined under stereomicroscope, light microscope and scanning electron microscope and micrographs of relevant details are included.

Physarum andinum, a new nivicolous species of myxomycete from the Patagonian Andes.

A new nivicolous species of Physarum was discovered during the study of myxomycetes in the Patagonian Andes of South America. It is described herein under the name Physarum andinum. The species is characterized by stalked sporophores or more rarely sessile sporocarps or short plasmodiocarps. The sporocarps are strikingly large, reaching 2.6 mm tall and 3 mm diam when open, and have a peridium with three layers, the internal layer being clearly visible and opening separately. Physarum andinum was found at five localities in Argentina as well as in herbarium material collected about 100 y ago in Chile. The new species is reminiscent of the non-nivicolous species Physarum brunneolum, but the latter forms smaller sporophores, has darker spores and the three layers of the peridium are adhered and open together. The characters of the new species were examined under stereomicroscope, light microscope and scanning electron microscope and micrographs of relevant details are included.

Microbotryum heliospermae, a new anther smut fungus parasitic on Heliosperma pusillum in the mountains of the European Alpine System.

The members of the smut genus Microbotryum are pathogens of a wide range of host plant species from nine dicotyledonous families. Within the genus, the species sporulating in anthers of Caryophyllaceae form a monophyletic group that in recent years attracted much interest in various biological studies. The phylogenetic framework developed for species delimitation within Microbotryum revealed that high level host-specificity is a major feature of most caryophyllaceous anther smuts. However, the great number of anther smut specimens on diverse host plant species reported worldwide has still not been included in phylogenetic analyses due to the inaccessibility of recently collected specimens, and thus many species remain still undiscovered. In this study, anther smut specimens on Heliosperma pusillum originating from all main mountain ranges of the European Alpine System were examined using partial rDNA sequence and/or morphological analyses. The investigation revealed that all specimens are morphologically uniform and phylogenetically represent a monophyletic lineage, sister to Microbotryum lagerheimii complex on Atocion rupestre/Silene lacera/Silene vulgaris/Viscaria vulgaris. This lineage cannot be attributed to any of the previously described species, and therefore the smut in anthers of H. pusillum is described and illustrated here as a new species, Microbotryum heliospermae. The species is known from subalpine zone of the Alps, the Carpathians, the Dinaric Alps, and the Pyrenees, inhabiting host plants growing in open spring communities or semihumid mountain meadows.

Rhizomarasmius epidryas (Physalacriaceae): phylogenetic placement of an arctic-alpine fungus with obligate saprobic affinity to Dryas spp.

As a part of a large-scale biogeographical study we examined the evolutionary relationships and taxonomic position of Marasmius epidryas, one of the most typical circumpolar arctic-alpine fungi, characterized by a specific, saprobic affinity to dead tissues of Dryas spp. A phylogenetic analysis based on nLSU and RPB2 DNA regions unequivocally indicated the phylogenetic placement of this species within the Physalacriaceae. The Bayesian MCMCMC analysis as well as other inference methods tested (ML, NJ) revealed a well supported affinity of M. epidryas to Rhizomarasmius pyrrhocephalus, type species of a recently circumscribed genus, Rhizomarasmius. As a consequence, based on these results, we introduce a new combination, Rhizomarasmius epidryas (Kühner ex A. Ronikier) A. Ronikier and M. Ronikier. Thus our results demonstrate that neither the traditional taxonomic placement of the fungus in genus Marasmius nor the recent transfer into genus Mycetinis are phylogenetically correct. In contrast they support the importance of the third lineage of the polyphyletic Marasmius s. l., having evolutionary links with taxa forming the Physalacriaceae clade of agaricoid fungi. In addition the lineage of Rhizomarasmius was confirmed to be closely related to the representatives of Gloiocephala, comprising small, often narrowly specialized saprobic species previously also classified within Marasmius s. l.

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.

Type study of Mycena phaeophylla reveals its conspecificity with M. clavata.

An extensive taxonomic study based on the type collection of Mycena phaeophylla revealed its conspecificity with M. clavata. Redescription of Kühner's original collections of M. phaeophylla is provided as well as a detailed description of M. clavata based on European material. A lectotype for M. phaeophylla is designated. Additional study on collections of M. speirea confirmed that this closely related species can be separated easily from M. clavata. New key to subsection Omphaliariae of section Hiemales is proposed.

Rediscovery of Alnicola cholea (Cortinariaceae): taxonomic revision and description of its mycorrhiza with Polygonum viviparum (Polygonaceae).

Alnicola cholea, a little-known species so far reported only from the two original localities in the French Alps, is redefined here based on revision of herbarium materials and studies of recent field collections. A detailed morphological and anatomical description of fruit bodies of Alnicola cholea, including a discussion on its taxonomic status and distribution data is provided. Due to the unique combination of characters of Alnicola cholea within the genus, a new monospecific section is introduced for this species: Alnicola sect. Cholea, sect. nov. Mycorrhizal symbiosis of A. cholea with an arcticalpine plant Polygonum viviparum was observed in the Tatra Mountains (Poland). A description of these mycorrhizae is given, providing first detailed data on an identified herbaceous plant mycorrhiza.

[Tricholoma equestre--animal toxicity study]. / Toksycznosc gaski zielonej--badania na modelu zwierzecym.

UNLABELLED: Animal toxicity study of Tricholoma equestre mushrooms stored for 12 months at (-)20 degrees C was performed using 30 male BALB/c mice. Three groups of 5 mice each were given suspension of T. equestre powder in water, boiled aqueous extract and chloroform-methanol extract dissolved in Miglyol 812 by gavage for three consecutive days. Mice in control groups were given water, Miglyol 812 and p-phenylenediamine (CAS 106-50-3). Creatine kinase activity was determined in serum collected 72 hours after the final dose. Mean activity of serum creatine kinase in mice treated with T. equestre powder, aqueous extract, chloroform-methanol extract and Miglyol 812 were 157 +/- 93, 129 +/- 30, 96 +/- 38, 111 +/- 66 U/L respectively and did not differ significantly from mean activity in mice which were given water (107 +/- 38 U/L). Mean serum creatine kinase activity in p-phenylenediamine group (265 +/- 63 U/L) was significantly higher than in group treated with water (p<0.01). CONCLUSION: Extracts of Tricholoma equestre mushrooms stored for 12 months at (-)20 degrees C did not cause rhabdomyolysis in male BALB/c mice.