DNA barcodes reliably differentiate between nivicolous species of Diderma (Myxomycetes, Amoebozoa) and reveal regional differences within Eurasia.

The nivicolous species of the genus Diderma are challenging to identify, and there are several competing views on their delimitation. We analyzed 102 accessions of nivicolous Diderma spp. that were sequenced for two or three unlinked genes to determine which of the current taxonomic treatments is better supported by molecular species delimitation methods. The results of a haplotype web analysis, Bayesian species delimitation under a multispecies coalescent model, and phylogenetic analyses on concatenated alignments support a splitting approach that distinguishes six taxa: Diderma alpinum, D. europaeum, D. kamchaticum, D. meyerae, D. microcarpum and D. niveum. The first two approaches also support the separation of Diderma alpinum into two species with allopatric distribution. An extended dataset of 800 specimens (mainly from Europe) that were barcoded with 18S rDNA revealed only barcode variants similar to those in the species characterized by the first data set, and showed an uneven distribution of these species in the Northern Hemisphere: Diderma microcarpum and D. alpinum were the only species found in all seven intensively sampled mountain regions. Partial 18S rDNA sequences serving as DNA barcodes provided clear signatures that allowed for unambiguous identification of the nivicolous Diderma spp., including two putative species in D. alpinum.

Lamproderma vietnamense: a new species of myxomycetes with reticulate spores from Phia Oac - Phia Dén National Park (northern Vietnam) supported by molecular phylogeny and morphological analysis.

A new species of Lamproderma (Myxomycetes), described herein as L. vietnamense, was recovered in the field on ground litter from mountain subtropical forests (Phia Oac - Phia Dén National Park) of northern Vietnam. Morphological details were examined by light and scanning electron microscopy. The species is characterized by a distinct and unique combination of morphological features, including a bright blue, shiny and very thin membranous peridium, a small dome-shaped columella, rigid, straight, branched, brown capillitial threads which gradually become pale at the periphery and finally colorless at the tips and small-meshed, banded-reticulate spores with 9-12 meshes across the spore diameter and solid walls without perforations 0.3-0.5 µm high. The stability of the taxonomic characters of L. vietnamense is supported by two well-developed collections found in 2018 and 2019. Partial sequences of three molecular markers (SSU, EF1α, COI) for both collections are identical. A two-gene phylogeny of the first two markers displays the two known accessions as a well-separated entity and indicates affinity of the new species with L. columbinum (the type taxon of the genus), L. violaceum, and several nivicolous Lamproderma species.

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.

Systematic revision of the Tubifera casparyi-T. dictyoderma complex: Resurrection of the genus Siphoptychium and introduction of the new genus Thecotubifera.

The genus Siphoptychium is resurrected on the basis of comparative morphology and phylogeny of partial nuc 18S rDNA (18S) and translation elongation factor 1-alpha (EF1A) nucleotide sequences. The genus is characterized by the firm upper surface of the pseudoaethalium, accreted but easily separable sporothecae, a tubular or fibrous columella, and spores with a reticulate ornamentation consisting of 7-9 meshes across the diameter. In addition to the currently known single species S. casparyi (= Tubifera casparyi), two new members of Siphoptychium are described: S. violaceum from coniferous forests of Europe, east Asia, and southeast Asia, and S. reticulatum from temperate and subarctic regions of North America and alpine regions of Europe. A second genus, Thecotubifera, is described to accommodate Tubifera dictyoderma. The fruiting body of this species is transitional between a pseudoaethalium and a true aethalium. It is covered by a contiguous membranous cortex formed by the fused tips of the sporothecae, a feature typical for aethalia. However, the inner portions of sporothecae remain discernible, a feature more typical for pseudoaethalia. Columellae of Th. dictyoderma are formed by perforated plates, and the spores have a reticulate ornamentation consisting of 2-5 meshes across the diameter. For Th. dictyoderma, we could confirm records only for tropical regions and Japan, whereas all studied European specimens, including those mentioned in current monographs, represent species of Siphoptychium.

A four year survey reveals a coherent pattern between occurrence of fruit bodies and soil amoebae populations for nivicolous myxomycetes.

Among soil-inhabiting protists, myxomycetes stand out by their macroscopic fructifications which have allowed studies on their ecology and distribution for more than two hundred years. One of the most distinct ecological guilds in myxomycetes are the nivicolous or "snowbank" myxomycete species, which produce fruit bodies at the edge of melting snowbanks in spring. Relationship between the occurrence of fructifications and myxamoebae remain unknown. In this study we used modern molecular techniques, by direct DNA amplification from soil extracts (NGS metabarcoding) to compare the distribution of soil-inhabiting myxamoebae found in 2016 with fructifications from the same sites collected over the course of four years (2013, 2015-17) along an elevational transect in the northern German Alps. A coherent community composition between fructification and soil myxamoebae, though with species-specific differences in relative abundance, was revealed. Although patterns varied among species, myxamoebae were found at both low and high elevations, whereas fruit bodies were mainly found at higher elevations, likely explained by the presence of a stable and long-lasting snow cover. In addition, a year to year comparison of fructification records support the hypothesis that the abundance of fructifications strongly depends on the onset of snowfall in the previous autumn and the soil temperature regime throughout the winter.

Genetic barcoding of dark-spored myxomycetes (Amoebozoa)-Identification, evaluation and application of a sequence similarity threshold for species differentiation in NGS studies.

Unicellular, eukaryotic organisms (protists) play a key role in soil food webs as major predators of microorganisms. However, due to the polyphyletic nature of protists, no single universal barcode can be established for this group, and the structure of many protistean communities remains unresolved. Plasmodial slime moulds (Myxogastria or Myxomycetes) stand out among protists by their formation of fruit bodies, which allow for a morphological species concept. By Sanger sequencing of a large collection of morphospecies, this study presents the largest database to date of dark-spored myxomycetes and evaluate a partial 18S SSU gene marker for species annotation. We identify and discuss the use of an intraspecific sequence similarity threshold of 99.1% for species differentiation (OTU picking) in environmental PCR studies (ePCR) and estimate a hidden diversity of putative species, exceeding those of described morphospecies by 99%. When applying the identified threshold to an ePCR data set (including sequences from both NGS and cloning), we find 64 OTUs of which 21.9% had a direct match (>99.1% similarity) to the database and the remaining had on average 90.2 ± 0.8% similarity to their best match, thus thought to represent undiscovered diversity of dark-spored myxomycetes.

Speciation in progress? A phylogeographic study among populations of Hemitrichia serpula (Myxomycetes).

Myxomycetes (plasmodial slime molds, Amoebozoa) are often perceived as widely distributed, confounding to the "everything is everywhere" hypothesis. To test if gene flow within these spore-dispersed protists is restricted by geographical barriers, we chose the widespread but morphologically unmistakable species Hemitrichia serpula for a phylogeographic study. Partial sequences from nuclear ribosomal RNA genes (SSU) revealed 40 ribotypes among 135 specimens, belonging to three major clades. Each clade is dominated by specimens from a certain region and by one of two morphological varieties which can be differentiated by SEM micrographs. Partial sequences of the protein elongation factor 1 alpha (EF1A) showed each clade to possess a unique combination of SSU and EF1A genotypes. This pattern is best explained assuming the existence of several putative biospecies dominating in a particular geographical region. However, occasional mismatches between molecular data and morphological characters, but as well heterogeneous SSU and heterozygous EF1A sequences, point to ongoing speciation. Environmental niche models suggest that the putative biospecies are rather restricted by geographical barriers than by macroecological conditions. Like other protists, myxomycetes seem to follow the moderate endemicity hypothesis and are in active speciation, which is most likely shaped by limited gene flow and reproductive isolation.

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.

Stable isotope composition (δ(13)C and δ(15)N values) of slime molds: placing bacterivorous soil protozoans in the food web context.

RATIONALE: Data on the bulk stable isotope composition of soil bacteria and bacterivorous soil animals are required to estimate the nutrient and energy fluxes via bacterial channels within detrital food webs. We measured the isotopic composition of slime molds (Myxogastria, Amoebozoa), a group of soil protozoans forming macroscopic spore-bearing fruiting bodies. An analysis of largely bacterivorous slime molds can provide information on the bulk stable isotope composition of soil bacteria. METHODS: Fruiting bodies of slime molds were collected in a monsoon tropical forest of Cat Tien National Park, Vietnam, and analyzed by continuous-flow isotope ratio mass spectrometry. Prior to stable isotope analysis, carbonates were removed from a subset of samples by acidification. To estimate the trophic position of slime molds, their δ(13) C and δ(15) N values were compared with those of plant debris, soil, microbial destructors (litter-decomposing, humus-decomposing, and ectomycorrhizal fungi) and members of higher trophic levels (oribatid mites, termites, predatory macroinvertebrates). RESULTS: Eight species of slime molds represented by at least three independent samples were 3-6‰ enriched in (13) C and (15) N relative to plant litter. A small but significant difference in the δ(13) C and δ(15) N values suggests that different species of myxomycetes can differ in feeding behavior. The slime molds were enriched in (15) N compared with litter-decomposing fungi, and depleted in (15) N compared with mycorrhizal or humus-decomposing fungi. Slime mold sporocarps and plasmodia largely overlapped with oribatid mites in the isotopic bi-plot, but were depleted in (15) N compared with predatory invertebrates and humiphagous termites. CONCLUSIONS: A comparison with reference groups of soil organisms suggests strong trophic links of slime molds to saprotrophic microorganisms which decompose plant litter, but not to humus-decomposing microorganisms or to mycorrhizal fungi. Under the assumption that slime molds are primarily feeding on bacteria, the isotopic similarity of slime molds and mycophagous soil animals indicates that saprotrophic soil bacteria and fungi are similar in bulk isotopic composition.

An improved kilogram-scale preparation of atorvastatin calcium.

BACKGROUND: If literature protocols are followed, conversion of an advanced ketal ester intermediate (available in kilogram quantities via a published Paal-Knorr synthesis) to cholesterol-lowering drug atorvastatin calcium is hampered by several process issues, particularly at the final stage where the hemi-calcium salt is obtained. RESULTS: We developed a high-yielding synthesis of atorvastatin calcium salt on 7 kg scale that affords >99.5% product purities by introducing the following key improvements: i. isolating the pure product of the ketal deprotection step as crystalline solid, and ii. using a convenient ethyl acetate extraction procedure to isolate the pure atorvastatin calcium at the ester hydrolysis and counter-ion exchange step. CONCLUSION: The convenient and operationally simple conversion of an advanced intermediate of atorvastatin to the clinically used hemi-calcium salt form of the drug that is superior to the methods obtainable from the literature is now available to facilitate the production of atorvastatin calcium on industrial scale. Graphical abstractStepwise ketal and tert-butyl ester group hydrolysis and a modified work-up protocol lead to a more convenient preparation of API-grade atorvastatin calcium.

A new species of Perichaena (Myxomycetes) with reticulate spores from southern Vietnam.

A new species of Perichaena, described herein as P. echinolophospora, was isolated in moist chamber cultures from substrate samples collected in the monsoon deciduous lowland tropical forests (Cat Tien National Park, Vinh Cuu Nature Reserve) and mixed montane tropical forests (Bi Dup-Nui Ba Nature Reserve) of southern Vietnam. Perichaena echinolophospora was recovered from ground litter, aerial litter and the bark of living trees. The morphology of representative specimens was examined by light and scanning electron microscopy, and micrographs of relevant details are provided. Spore ornamentation consists of a regular reticulum with large meshes, 3-4 µm in wide and limited by denticulate ridges, 1.7-2.2 µm tall, and clusters of small warts and rods densely distributed over the spore surface within the meshes of the reticulum and visible only by SEM. The stability of the taxonomic characters of the species was confirmed by several collections obtained during three field seasons.

Description, culture and phylogenetic position of a new xerotolerant species of Physarum.

A new widespread myxomycete species, Physarum pseudonotabile, inhabiting the arid regions of the Eurasia, South and North America is described and illustrated. Tentatively assigned to Ph. notabile T. Macbr., a phylogeny based on the small ribosomal subunit (SSU) and elongation factor 1 alpha (EF1a) genes placed the new species in a clade far from Ph. notabile. Ph. pseudonotabile was found to be frequent in surveys based on the moist chamber culture technique with samples of litter, bark and herbivore dung collected in dry steppe and deserts of the Caspian lowland (Russia), Kazakhstan, Mongolia, China, Spain, Argentina and USA. The main morphological difference between Ph. pseudonotabile and Ph. notabile lies in spore ornamentation. Spores of the former species display irregularly distributed verrucae, whereas the latter species possesses spores with dense and regularly arranged spinulae. In addition, the ecological preferences of the two species differ. Ph. pseudonotabile inhabits the bark of living plants and ground litter in arid regions, whereas Ph. notabile is found on coarse woody debris in boreal and temperate forests. Although the new species appears to be closest to Ph. notabile morphologically, the phylogenetic analysis reveals Ph. pusillum and Ph. nivale as the closest relatives. In addition, the molecular investigations revealed a considerable amount of hidden diversity within species of Physarum with gray lime flakes. Currently we have only sufficient material to assess the morphological variation of Ph. pseudonotabile but expect that more taxa within this clade may emerge within studies combining morphological and molecular analyses.

A new species of Trichia from Australia.

A new species of Trichia (myxomycetes) was collected during surveys for myxomycetes carried out in Nothofagus cunninghamii forests in western Tasmania in May 2008 and a similar survey carried out in a N. morrei forest in New South Wales in May 2009. This new species, T. brimsiorum, is described and illustrated. It resembles T. decipiens in overall shape and size of the sporocarps but has smaller spores and the ornamentation of the capillitium is different.

Genetic structure of two protist species (Myxogastria, Amoebozoa) suggests asexual reproduction in sexual Amoebae.

Plasmodial slime molds (Myxogastria or Myxomycetes) are common and widespread unicellular organisms that are commonly assumed to have a sexual life cycle culminating with the formation of often macroscopic fruiting bodies that efficiently disseminate spores. However, laboratory studies based on mating compatibility revealed the coexistence of asexual as well as sexual strains. To test this hypothesis in natural populations, we investigated the genetic variability of two species of the genus Lamproderma. Detailed ecological relevés were carried out in 2007 and 2009 in several deep ravines in the Elbsandsteingebirge (Saxony, south-eastern Germany). Morphological characters of 93 specimens of Lamproderma were recorded and genetic analyses, based on the small subunit ribosomal gene, the internal transcribed spacer 1 and partial elongation factor 1α sequences were carried out for 52 specimens. Genetic analyses showed the existence of two major clades, each composed of several discrete lineages. Most of these lineages were composed of several identical sequences (SSU, ITS 1 and EF-1α) which is explained best by an asexual mode of reproduction. Detrended Correspondence Analysis of morphological characters revealed two morphospecies that corresponded to the two major clades, except for one genotype (Lc6), thus challenging the morphospecies concept. Genetic patterns were not related to the geographical distribution: specimens belonging to the same genotype were found in distinct ravines, suggesting effective long-distance dispersal via spores, except for the Lc6 genotype which was found only in one ravine. Implications for the morphological and biological species concept are discussed.

Two new species of Perichaena (Myxomycetes) from arid areas of Russia and Kazakhstan.

Two new myxomycete species from dry steppe and desert communities of the Caspian Lowland (Russia) and central Kazakhstan are described and illustrated. They are placed tentatively within genus Perichaena, which does include species with a reduced capillitium and single-layered peridium. Both species were found repeatedly in moist chamber cultures; P. heterospinispora appeared on leaf litter and twigs, whereas P. polygonospora occurred on leaf litter and weathered dung of rodents. Both species have spore ornamentation that is unique for members of genera Licea and Perichaena. The spore ornamentation of the first species includes scattered large, pyramid-like spines 0.9-1.2 microm high that sometimes have enlarged ends. Among these spines the spore surface is covered by evenly and densely distributed warts that are visible only by SEM. The second species is characterized by angular spores with a coarse network of rounded ridges. The areas among these ridges bear scattered composite warts 0.3-0.5 microm high that sometimes coalesce to form clusters but more often are distributed evenly and densely and are visible only by SEM. The stability of the taxonomic characters of both species was confirmed by several collections from different regions obtained in 2 y. The morphology of the fructifications of the two myxomycetes was examined with both scanning electron and light microscopy, and micrographs of all relevant features are presented.