Vasilii Shapkin

and 7 more

The nuclear ribosomal DNA Internal Transcribed Spacer (ITS) region is used as universal fungal barcode marker, but it is often missing significant DNA barcoding gap between sister taxa. Here we tested reliability of protein coding low-copy genes as alternative markers. Mock communities of three unrelated agaric genera (Dermoloma, Hodophilus and Russula) representing lineages of closely related species were sequenced by Illumina platform targeting ITS1, ITS2, the second largest subunit of RNA polymerase II gene (rpb2) and the transcription elongation factor 1-alpha gene (ef1-α) regions. The representation of species and their relative abundances were similar in all tested barcode regions, despite lower copy number in protein coding markers. ITS1 and ITS2 required more sophisticated sequence filtering because they produced a high number of chimeric sequences requiring reference-based chimera removal and had higher number of sequence variants per species. Clustering of filtered ITS sequences showed in average higher number of correctly clustered units at best fitted similarity thresholds, but these thresholds were very different among genera. Best fitted thresholds of low-copy markers were more consistent among genera but species resolution was frequently missing due to low intraspecific variability. At some thresholds we observed multiple species lumped together and, at the same time, species split in multiple partial clusters, which should be taken into consideration when assessing best clustering thresholds and taxonomic identity of clusters. For best taxonomic resolution and better species detection, we recommend to combine different markers and to apply additional reference-based sorting of clusters.

Tereza Veselská

and 8 more

Ips typographus, the most serious pest of spruce forests in Europe, is associated with microorganisms facilitating its invasion and development inside spruce tissues. Despite the importance of I. typographus, little is known about its core gut microbiome. Hereby, we describe the composition of bacterial and fungal microbiomes throughout I. typographus life cycle in spring and summer generations. We used cultivation technique and molecular identification in combination with DNA and RNA metabarcoding to achieve deep inside into the beetle’s microbiome composition and structure. As it is not known whether microbiome forms stable communities inside the beetle’s gut, we observed gut epithelium for biofilm formation with Transmission Electron Microscopy. Cultivation technique together with DNA and RNA metabarcoding indicated similar dominant taxa. The bacterial community belongs almost exclusively to the phylum Proteobacteria (newly Pseudomonadota) and the most common orders and genera are Enterobacteriales (Erwinia and Serratia), Pseudomonadales (Pseudomonas) and Xanthomonadales (Pseudoxanthomonas, Stenotrophomonas). Yeasts (Saccharomycetes) highly dominated the fungal microbiome, followed by Sordariomycetes represented mainly by Ophiostoma bicolor and Endoconidiophora polonica. The most common yeasts were Wickerhamomyces bisporus, Kuraishia molischiana, Nakazawaea ambrosiae, Yamadazyma spp. and Cyberlindnera sp. The proportions of the dominant taxa belonging to the core microbiome of I. typographus change throughout its life cycle and generations. We did not observe any biofilm formation on gut epithelium, which suggests that microbial cells pass through the beetle’s gut with chyme. We propose that species belonging to the core microbiome has similar functions and alternate in the I. typographus ecosystem depending on environmental conditions.