Basidiobolus omanensis sp. nov. Causing Angioinvasive Abdominal Basidiobolomycosis

Human infectious fungal diseases are increasing, despite improved hygienic conditions. We present a case of gastrointestinal basidiobolomycosis (GIB) in a 20-year-old male with a history of progressively worsening abdominal pain. The causative agent was identified as a novel Basidiobolus species. Validation of its novelty was established by analysis of the partial ribosomal operon of two isolates from different organs. Phylogeny of ITS and LSU rRNA showed that these isolates belonged to the genus Basidiobolus, positioned closely to B. heterosporus and B. minor. Morphological and physiological data supported the identity of the species, which was named Basidiobolus omanensis, with CBS 146281 as the holotype. The strains showed high minimum inhibitory concentrations (MICs) to fluconazole (>64 µg/mL), itraconazole and voriconazole (>16 µg/mL), anidulafungin and micafungin (>16 µg/mL), but had a low MIC to amphotericin B (1 µg/mL). The pathogenic role of B. omanensis in gastrointestinal disease is discussed. We highlight the crucial role of molecular identification of these rarely encountered opportunistic fungi.

Global distribution and richness of Armillaria species inferred from public databases and amplicon sequencing datasets

Armillaria is a globally distributed fungal genus most notably recognized as economically important plant pathogens that are found predominantly in forest and agronomic systems. Armillaria has more recently received attention for ecologically diverse roles as woody plant decomposers and as mycorrhizal symbionts with specific plants. Previous phylogenetic analyses suggest that around 50 species are recognized globally. Despite this previous work, no studies have analyzed the global species richness and distribution of the genus using data derived from fungal community sequencing datasets. In order to assess the global diversity and species richness of Armillaria, we mined publicly available sequencing datasets derived from numerous primer pairs for the ribosomal operon. Our estimates reveal that species richness ranges from 50 to 60 species, depending on which marker is used. Eastern Asia represents the biogeographic region with the highest species richness. We also assess the overlap of species across geographic regions and propose some hypotheses regarding the drivers of variability in species diversity and richness between different biogeographic regions.

Culicidae-centric metabarcoding through targeted use of D2 ribosomal DNA primers

A practical limitation to many metabarcoding initiatives is that sampling methods tend to collect many non-target taxa, which become “amplicon noise” that can saturate Next Generation Sequencing results and lead to both financial and resource inefficiencies. An available molecular tool that can significantly decrease these non-target amplicons and decrease the need for pre-DNA-extraction sorting of bycatch is the design of PCR primers tailored to the taxa under investigation. We assessed whether the D2 extension segment of the 28S ribosomal operon can limit this shortcoming within the context of mosquito (Culicidae) monitoring. We designed PCR primers that are fully conserved across mosquitos and exclude from amplification most other taxa likely to be collected with current sampling apparatuses. We show that, given enough sequencing depth, D2 is an effective marker for the detection of mosquito sequences within mock genomic DNA pools. As few as 3,050 quality-filtered Illumina reads were able to recover all 17 species in a bulk pool containing as little as 0.2% of constituent DNA from single taxa. We also mixed these mosquito DNA pools with high concentrations of non-Culicidae bycatch DNA and show that the component mosquito species are generally still recoverable and faithful to their original relative frequencies. Finally, we show that there is little loss of fidelity in abundance parameters when pools from degraded DNA samples were sequenced using the D2 primers.

Characterizing the ribosomal tandem repeat and its utility as a DNA barcode in lichen-forming fungi

Background Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing. Results We estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of this complex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, intergenic spacer region, and some intronic regions, independently; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny. Conclusions The results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.

Characterizing the ribosomal tandem repeat and its utility as a DNA barcode in lichen-forming fungi

Background: Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing. Results: We estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of thiscomplex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, intergenic spacer region, and some intronic regions, independently; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny. Conclusions: The results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.

Characterizing the ribosomal tandem repeat and its utility as a DNA barcode in lichen-forming fungi

Background: Regions within the nuclear ribosomal operon are a major tool for inferring evolutionary relationships and investigating diversity in fungi. In spite of the prevalent use of ribosomal markers in fungal research, central features of nuclear ribosomal DNA (nrDNA) evolution are poorly characterized for fungi in general, including lichenized fungi. The internal transcribed spacer (ITS) region of the nrDNA has been adopted as the primary DNA barcode identification marker for fungi. However, little is known about intragenomic variation in the nrDNA in symbiotic fungi. In order to better understand evolution of nrDNA and the utility of the ITS region for barcode identification of lichen-forming fungal species, we generated nearly complete nuclear ribosomal operon sequences from nine species in the Rhizoplaca melanophthalma species complex using short reads from high-throughput sequencing. Results: We estimated copy numbers for the nrDNA operon, ranging from nine to 48 copies for members of thiscomplex, and found low levels of intragenomic variation in the standard barcode region (ITS). Monophyly of currently described species in this complex was supported in phylogenetic inferences based on the ITS, 28S, IGS, and some intronic regions; however, a phylogenetic inference based on the 18S provided much lower resolution. Phylogenetic analysis of concatenated ITS and intergenic spacer sequence data generated from 496 specimens collected worldwide revealed previously unrecognized lineages in the nrDNA phylogeny. Conclusions: The results from our study support the general assumption that the ITS region of the nrDNA is an effective barcoding marker for fungi. For the R. melanophthalma group, the limited amount of potential intragenomic variability in the ITS region did not correspond to fixed diagnostic nucleotide position characters separating taxa within this species complex. Previously unrecognized lineages inferred from ITS sequence data may represent undescribed species-level lineages or reflect uncharacterized aspects of nrDNA evolution in the R. melanophthalma species complex.

A genome-skimmed phylogeny of a widespread bryozoan family, Adeonidae

Background Understanding the phylogenetic relationships among species is one of the main goals of systematic biology. Simultaneously, credible phylogenetic hypotheses are often the first requirement for unveiling the evolutionary history of traits and for modelling macroevolutionary processes. However, many non-model taxa have not yet been sequenced to an extent such that statistically well-supported molecular phylogenies can be constructed for these purposes. Here, we use a genome-skimming approach to extract sequence information for 15 mitochondrial and 2 ribosomal operon genes from the cheilostome bryozoan family, Adeonidae, Busk, 1884, whose current systematics is based purely on morphological traits. The members of the Adeonidae are, like all cheilostome bryozoans, benthic, colonial, marine organisms. Adeonids are also geographically widely-distributed, often locally common, and are sometimes important habitat-builders. Results We successfully genome-skimmed 35 adeonid colonies representing 6 genera (Adeona, Adeonellopsis, Bracebridgia, Adeonella, Laminopora and Cucullipora). We also contributed 16 new, circularised mitochondrial genomes to the eight previously published for cheilostome bryozoans. Using the aforementioned mitochondrial and ribosomal genes, we inferred the relationships among these 35 samples. Contrary to some previous suggestions, the Adeonidae is a robustly supported monophyletic clade. However, the genera Adeonella and Laminopora are in need of revision: Adeonella is polyphyletic and Laminopora paraphyletically forms a clade with some Adeonella species. Additionally, we assign a sequence clustering identity using cox1 barcoding region of 99% at the species and 83% at the genus level. Conclusions We provide sequence data, obtained via genome-skimming, that greatly increases the resolution of the phylogenetic relationships within the adeonids. We present a highly-supported topology based on 17 genes and substantially increase availability of circularised cheilostome mitochondrial genomes, and highlight how we can extend our pipeline to other bryozoans.

Taxonomic resolution of the ribosomal RNA operon in bacteria: implications for its use with long-read sequencing

DNA barcoding through the use of amplified regions of the ribosomal operon, such as the 16S gene, is a routine method to gain an overview of the microbial taxonomic diversity within a sample without the need to isolate and culture the microbes present. However, bacterial cells usually have multiple copies of this ribosomal operon, and choosing the ‘wrong’ copy could provide a misleading species classification. While this presents less of a problem for well-characterized organisms with large sequence databases to interrogate, it is a significant challenge for lesser known organisms with unknown copy number and diversity. Using the entire length of the ribosomal operon, which encompasses the 16S, 23S, 5S and internal transcribed spacer regions, should provide greater taxonomic resolution but has not been well explored. Here, we use publicly available reference genomes and explore the theoretical boundaries when using concatenated genes and the full-length ribosomal operons, which has been made possible by the development and uptake of long-read sequencing technologies. We quantify the issues of both copy choice and operon length in a phylogenetic context to demonstrate that longer regions improve the phylogenetic signal while maintaining taxonomic accuracy.