Internal Transcribed Spacer 1 Secondary Structure Analysis Reveals a Common Core throughout the Anaerobic Fungi (Neocallimastigomycota)

Abstract

The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

Buccal Swabbing as a Noninvasive Method To Determine Bacterial, Archaeal, and Eukaryotic Microbial Community Structures in the Rumen

“…Ciliate protozoal sequences were clustered using the prefix-suffix method (22). Representative OTUs were subjected to BLAST searches against a newly assembled BLAST database composed of the following databases: a modified Greengenes gg_13_5 database, which was depleted of all sequences with taxonomy assignments beginning with "k__Archaea" (26); RIM-DB (25); and databases for intestinal ciliate protozoa (27) and anaerobic fungi (28). Relative abundance tables were generated at the species level (bacteria and archaea) or genus level (ciliate protozoa).…”

Buwchfawromyces eastonii gen. nov., sp. nov.: a new anaerobic fungus (Neocallimastigomycota) isolated from buffalo faeces

“…material 4) also fall into the Buwchfawromyces clade. This clade was also identified (and denoted as clade SK2) in the recent study by Koetschan et al (2014), who were able to create a reliable phylogeny by using predictors of ITS1 folding to decrease the effects of gaps in anaerobic fungal ITS1 alignments.…”

“…Clades corresponding to the known genera, the new Buwchfawromyces clade and also the 'polycephalus' clade are labelled. Codes in brackets indicate the novel clades identified by Koetschan et al (2014). in order to improve the quality of the alignments (fewer gaps). Additionally, 'environmental' sequences obtained from clone library studies, (mostly labelled as "uncultured Neocallimastigales") were also included, as recommended by Nilsson et al (2011).…”

“…It was originally isolated and described from buffalo in Taiwan (isolate W-33; (Chen et al 2002)) and has since been reported from India (isolate CTS-47 from zebra faeces; GenBank EU330178); this clade was denoted DT1 (Fig. 3) by Koetschan et al (2014). Given that 'A.…”

Technical note: use of internal transcribed spacer for ruminal yeast identification in dairy cows

“…ITS is a noncoding ribosomal DNA (rDNA) spacer region located between the 18S and 28S ribosomal RNA (rRNA) genes (Sirohi et al, 2013), and comprises ITS1/ITS2 intergenic sequences with highly conserved 5.8 rRNA in between (Zhang et al, 2015). These regions were confirmed to be the most suitable marker genes for all classes of fungi providing complementary phylogenetic information (Koetschan et al, 2014), and even useful for the detection and identification of fungal pathogens (Iwen et al, 2005). Moreover, ITS is a DNA region with high variation among closely related species due to its fast rate of evolution (Schoch et al, 2012) providing a molecular basis for determining phylogenetic relationships (Hayashi et al, 2013).…”