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.

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.

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.

Two independent genetic markers support separation of the myxomycete Lycogala epidendrum into numerous biological species.

Lycogala epidendrum is one of the most widely known myxomycete species and the first-ever discovered representative of this group. Using 687 original DNA sequences from 330 herbarium specimens from Europe, Asia, North and Central America, and Australia, we constructed the first detailed phylogenies of the genus Lycogala, based on two independently inherited genetic markers, the ribosome small subunit 18S rRNA nuclear gene (18S rDNA) and the mitochondrial cytochrome oxidase subunit I gene (COI). In both phylogenies, L. epidendrum appeared to be a polyphyletic group, represented by numerous clades. The four other recognized species of the genus (L. confusum, L. conicum, L. exiguum, and L. flavofuscum) are scattered between branches corresponding to L. epidendrum. A barcode gap analysis revealed 60 18S rDNA phylogroups of L. epidendrum, which are distant from each other not less than from other species of the genus Lycogala. For 18 of these phylogroups with both 18S rDNA and COI sequences available, recombination patterns were analyzed to test for reproductive isolation. In contrast to the results of a simulation assuming panmixis, no crossing between ribosomal and mitochondrial phylogroups was found, thus allowing the conclusion that all tested phylogroups represent biospecies. More than one third (39.6%) of the studied specimens share a single 18S rDNA phylogroup, which we consider to be L. epidendrum s. str. This group displays the broadest geographic distribution and the highest intraspecific genetic variability. Nearly all (93.3%) of the remaining non-singleton 18S rDNA phylogroups are restricted to certain continents or even regions. At the same time, various reproductively isolated phylogroups occur sympatric at a given location.

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.

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.

Strategies for Intraspecific Genotyping of Duckweed: Comparison of Five Orthogonal Methods Applied to the Giant Duckweed Spirodela polyrhiza.

The predominantly vegetative propagating duckweeds are of growing commercial interest. Since clonal accessions within a respective species can vary considerably with respect to their physiological as well as biochemical traits, it is critical to be able to track the clones of species of interest after their characterization. Here, we compared the efficacy of five different genotyping methods for Spirodela polyrhiza, a species with very low intraspecific sequence variations, including polymorphic NB-ARC-related loci, tubulin-gene-based polymorphism (TBP), simple sequence repeat variations (SSR), multiplexed ISSR genotyping by sequencing (MIG-seq), and low-coverage, reduced-representation genome sequencing (GBS). Four of the five approaches could distinguish 20 to 22 genotypes out of the 23 investigated clones, while TBP resolved just seven genotypes. The choice for a particular method for intraspecific genotyping can depend on the research question and the project budget, while the combination of orthogonal methods may increase the confidence and resolution for the results obtained.

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.

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.