Simultaneous Metabarcoding and Quantification of Neocallimastigomycetes from Environmental Samples: Insights into Community Composition and Novel Lineages

Abstract

Anaerobic fungi from the herbivore digestive tract (Neocallimastigomycetes) are primary lignocellulose modifiers and hold promise for biotechnological applications. Their molecular detection is currently difficult due to the non-specificity of published primer pairs, which impairs evolutionary and ecological research with environmental samples. We developed and validated a Neocallimastigomycetes-specific PCR primer pair targeting the D2 region of the ribosomal large subunit suitable for screening, quantifying, and sequencing. We evaluated this primer pair in silico on sequences from all known genera, in vitro with pure cultures covering 16 of the 20 known genera, and on environmental samples with highly diverse microbiomes. The amplified region allowed phylogenetic differentiation of all known genera and most species. The amplicon is about 350 bp long, suitable for short-read high-throughput sequencing as well as qPCR assays. Sequencing of herbivore fecal samples verified the specificity of the primer pair and recovered highly diverse and so far unknown anaerobic gut fungal taxa. As the chosen barcoding region can be easily aligned and is taxonomically informative, the sequences can be used for classification and phylogenetic inferences. Several new Neocallimastigomycetes clades were obtained, some of which represent putative novel lineages such as a clade from feces of the rodent Dolichotis patagonum (mara).

Effect of Growth Media on the Diversity of Neocallimastigomycetes from Non-Rumen Habitats

“…The recently developed Neocallimastigomycetes -specific primer pair [ 8 ] used in this study generated 775,703 reads—76% of filtered reads were identified as unique ASVs ( Table S2 ). The majority of ASVs were affiliated with Khoyollomyces ramosus (28.9%), Neocallimastix spp.…”

“…A total of 200 mg of washed sample was used for the total genomic DNA extraction using a GenElute Soil DNA Isolation Kit (Sigma, St. Louis, MO, USA), following the manufacturer’s instructions. The polymerase chain reactions (PCR) were performed using the AGF-LSU-EnvS primer pair amplifying the D2 LSU region according to Young et al [ 8 ]. The generated amplicons, along with the attached forward Illumina adapter: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ (33 bp) and a reverse Illumina adapter: 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′ (34 bp) were sent for Miseq Illumina sequencing (2 × 300 bp chemistry) to an external service provided by the Core Facility Microbiome at ZIEL—Institute for Food & Health, Technical University of Munich, Germany.…”

“…Taxonomic classifications were carried out using the native implementation of the QIIME2 pipeline called q2-feature-classifier. Since there is a significant lack of reference sequences for AF, we compiled a robust in-house 28S rDNA (LSU) sequence database of AF [ 8 ]. These LSU reference sequences include novel AF species and genera that are published by Young et al [ 8 ].…”

“…Since there is a significant lack of reference sequences for AF, we compiled a robust in-house 28S rDNA (LSU) sequence database of AF [ 8 ]. These LSU reference sequences include novel AF species and genera that are published by Young et al [ 8 ]. Using this customized reference database, a high-resolution classifier model was trained for taxonomic classification.…”

“…For several decades, the taxonomy of AF was mainly based on six genera—namely, Neocallimastix , Caecomyces , Orpinomyces , Piromyces , Anaeromyces, and Cyllamyces [ 2 , 3 ]. Recently, the diversity of AF is gaining a new shape—a total of 20 genera have been named within the phylum Neocallimastigomycota , comprising 36 species, of which half of the genera were described in the last five years [ 4 , 5 ], and many more still need to be discovered [ 1 , 6 , 7 , 8 ]. Several reasons explain the remaining gap of knowledge on AF diversity and their propagation, most prominently the relatively short history of AF isolation, improper sampling and isolation techniques, and insufficient sampling sources [ 9 ].…”