Current exploration of the ecology of soil fungal and bacterial communities and microbe-catalyzed processes in soils largely rely on community composition analysis using next-generation-sequencing of PCR amplicons (1). Typically, the relative abundance of individual members of microbial communities are derived from the analyses of 16S rRNA region of prokaryotic microorganisms and 18S rRNA or internal transcribed spacer (ITS) region of the rDNA for fungi and other microeukaryots. The analysis of fungal ITS sequences is helpful tool for molecular systematics at the species level, and even within species, but the quantitative information on the relative abundance of individual taxa is skewed due to the presence of multiple rDNA gene copies per genome, ranging from 10 to 200 (2). On the other hand, it was demonstrated that there is a group of genes like the elongation factor-1 alpha (tef1) or RNA polymerase II second largest subunit (rpb2) that are consistently present in one copy per fungal genome and exhibit sufficient variation to be used for phylogenetic analysis and taxonomic assignment (3). The use of such genes offers the possibility to directly count fungal genomes and improve the knowledge on the relative importance of individual taxa of fungi in the environmental processes. Here we demonstrate that the amount of ITS copies per nanogram DNA shows high variation among soil basidiomycetes and even closely related species largely differ in this respect. We also demonstrate that the use of the rpb2 gene is applicable for analysis of soil fungal communities and that the data derived using this molecular marker are largely different from those based on the amplicon sequencing of the ITS. Although the phylogeneti discriminative power of the rpb2 gene is limited, it still offers a suitable tool to infer fungal taxonomy at least on the level of families.

Although sexual reproduction is ubiquitous throughout nature, the molecular machinery behind it has been repeatedly disrupted during evolution, leading to the emergence of asexual lineages in all eukaryotic phyla. Despite intensive research, little is known about what causes the switch from sexual reproduction to asexuality.Interspecific hybridization is one of the candidate explanations, but the reasons for the apparent association between hybridization and asexuality remain unclear. In this study, we combined cross-breeding experiments with population genetic and phylogenomic approaches to reveal the history of speciation and asexuality evolution in European spined loaches (Cobitis). Contemporary species readily hybridize in hybrid zones, but produce infertile males and fertile but clonally reproducing females that cannot mediate introgressions. However, our analysis of exome data indicates that intensive gene flow between species has occurred in the past. Crossings among species with various genetic distances showed that, while distantly related species produced asexual females and sterile males, closely related species produce sexually reproducing hybrids of both sexes. Our results suggest that hybridization leads to sexual hybrids at the initial stages of speciation, but as the species diverge further, the gradual accumulation of reproductive incompatibilities between species could distort their gametogenesis towards asexuality. Interestingly, comparative analysis of published data revealed that hybrid asexuality generally evolves at lower genetic divergences than hybrid sterility or inviability. Given that hybrid asexuality effectively restricts gene flow, it may establish a primary reproductive barrier earlier during diversification than other "classical" forms of postzygotic incompatibilities. Hybrid asexuality may thus indirectly contribute to the speciation process. K E Y W O R D Sbalance hypothesis, coalescence, evolution of asexuality, hybridization, phylogeography,

BackgroundProbiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD.Methodology/Principal FindingsThe preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4+FoxP3+ regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4+FoxP3+ regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer's patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway.Conclusion/SignificanceOur study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.