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.

High Environmentally Induced Plasticity in Spore Size and Numbers of Nuclei per Spore in Physarum albescens (Myxomycetes).

Spore size enables dispersal in plasmodial slime molds (Myxomycetes) and is an important taxonomic character. We recorded size and the number of nuclei per spore for 39 specimens (colonies of 50-1000 sporocarps) of the nivicolous myxomycete Physarum albescens, a morphologically defined taxon with several biological species. For each colony, three sporocarps were analyzed from the same spore mount under brightfield and DAPI-fluorescence, recording ca. 14,000 spores per item. Diagrams for spore size distribution showed narrow peaks of mostly uninucleate spores. Size was highly variable within morphospecies (10.6-13.5 µm, 11-13%), biospecies (3-13%), even within spatially separated colonies of one clone (ca. 8%); but fairly constant for a colony (mean variation 0.4 µm, ca. 1.5%). ANOVA explains most of this variation by the factor locality (within all colonies: 32.7%; within a region: 21.4%), less by biospecies (13.5%), whereas the contribution of intra-colony variation was negligible (<0.1%). Two rare aberrations occur: 1) multinucleate spores and 2) oversized spores with a double or triple volume of normal spores. Both are not related to each other or limited to certain biospecies. Spore size shows high phenotypic plasticity, but the low variation within a colony points to a strong genetic background.

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.

A workflow for low-cost automated image analysis of myxomycete spore numbers, size and shape.

Measuring spore size is a standard method for the description of fungal taxa, but in manual microscopic analyses the number of spores that can be measured and information on their morphological traits are typically limited. To overcome this weakness we present a method to analyze the size and shape of large numbers of spherical bodies, such as spores or pollen, by using inexpensive equipment. A spore suspension mounted on a slide is treated with a low-cost, high-vibration device to distribute spores uniformly in a single layer without overlap. Subsequently, 10,000 to 50,000 objects per slide are measured by automated image analysis. The workflow involves (1) slide preparation, (2) automated image acquisition by light microscopy, (3) filtering to separate high-density clusters, (4) image segmentation by applying a machine learning software, Waikato Environment for Knowledge Analysis (WEKA), and (5) statistical evaluation of the results. The technique produced consistent results and compared favorably with manual measurements in terms of precision. Moreover, measuring spore size distribution yields information not obtained by manual microscopic analyses, as shown for the myxomycete Physarum albescens. The exact size distribution of spores revealed irregularities in spore formation resulting from the influence of environmental conditions on spore maturation. A comparison of the spore size distribution within and between sporocarp colonies showed large environmental and likely genetic variation. In addition, the comparison identified specimens with spores roughly twice the normal size. The successful implementation of the presented method for analyzing myxomycete spores also suggests potential for other applications.

Feeding study for the mycotoxin zearalenone in yellow mealworm (Tenebrio molitor) larvae-investigation of biological impact and metabolic conversion.

Edible insects as additional food and/or feed source may represent one important component to solve the problem of food security for a growing human population. Especially for covering the rising demand for protein of animal origin, seven insect species currently allowed as feed constituents in the European Union are gaining more interest. However, before considering insects such as yellow mealworm larvae (Tenebrio molitor) as suitable for, e.g. human consumption, the possible presence and accumulation of contaminants must be elucidated. The present work investigates the effects of the mycotoxin zearalenone (ZEN) and its metabolites on insect larvae. Seven different diets were prepared: toxin-free control, spiked and artificially contaminated (both containing approx.500 µg/kg and approx. 2000 µg/kg of ZEN) as well as two naturally contaminated diets (600 µg/kg and 900 µg/kg ZEN). The diets were used in a multiple-week feeding trial using T. molitor larvae as model insects. The amount of ZEN and its metabolites in the feed, larvae and the residue were measured by HPLC-MS/MS. A significantly enhanced individual larval weight was found for the insects fed on the naturally contaminated diets compared to the other feeding groups after 8 weeks of exposure. No ZEN or ZEN metabolites were detected in the T. molitor larvae after harvest. However, ZEN, α- and ß-stereoisomers of zearalenol were found in the residue samples indicating an intense metabolism of ZEN in the larvae. No further ZEN metabolites could be detected in any sample. Thus, ZEN is not retained to any significant amount in T. molitor larvae.