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.

Methylobacterium bullatum sp. nov., a methylotrophic bacterium isolated from Funaria hygrometrica.

A novel, pink-pigmented aerobic, facultatively methylotrophic bacterial strain (F3.2(T)) isolated from the phyllosphere of Funaria hygrometrica, was analyzed using a polyphasic approach. Cells were Gram-negative, motile rods, strictly aerobic and non-spore-forming and exhibited surface structures varying in quantity, distribution and morphology. The isolate grew at 10-33°C over a pH range of 5.5-8.0 and in the presence of less than 1.0% NaCl. Strain F3.2(T) shared less than 70% DNA-DNA binding to the next type strain of the genus Methylobacterium (M. adhaesivum DSM 17169(T)). In addition to the major cellular fatty acid C(18:1)ω7c (81.7%), present in all Methylobacterium species (and also members of the genus Alphaproteobacteria), a high value (11.7%) of the fatty acids (summed feature) C(16:1)ω7c and/or iso-C(15:0)2OH was determined. Phylogenetic analyses based on 16S rDNA and methanol dehydrogenase gene sequences, DNA-DNA hybridization values, chemotaxonomic and phenotypic characteristics indicate that the strain F3.2(T) represents a novel species within the genus Methylobacterium. We propose the name Methylobacterium bullatum sp. nov. for this species. The type strain is the strain F3.2(T) (DSM 21893(T)=LMG 24788(T)).