Full ribosomal operon sequencing finally cracks the family tree of anaerobic gut fungi

Anaerobic gut fungi are among the strangest and most important microbes on Earth. They live in the oxygen-free stomachs of cattle, sheep, elephants and other herbivores, where they shred tough plant fibers and even team up with methane-producing archaea to shape the greenhouse emissions of their hosts. Yet despite decades of study, scientists have struggled to draw a reliable family tree for this entire fungal phylum, known as Neocallimastigomycota. A new study published in IMA Fungus now changes that, using long-read sequencing to capture the complete ribosomal RNA operon of 156 isolates and revealing genetic markers that finally separate lineages that have resisted classification for twenty years.

The problem has always been that anaerobic gut fungi look frustratingly alike under the microscope. Taxonomists distinguish genera by whether the thallus is filamentous or bulbous, whether zoospores carry one or many flagella, and whether the rhizoidal system is monocentric or polycentric. But these traits can overlap across species and even across genera, and distinctive structures can disappear entirely after repeated subculturing in the laboratory. DNA sequencing has therefore become the primary basis for species-level classification, yet the markers most commonly used, the internal transcribed spacer regions and the D1/D2 domains of the large ribosomal subunit, often fail to resolve relationships between closely related lineages.

A striking example involves the genera Caecomyces and Cyllamyces. Both produce bulbous thalli with spherical holdfasts and have been isolated from the same hosts, including cattle, sheep and water buffalo. The main morphological distinction is that Cyllamyces exhibits polycentric thalli while Caecomyces presents monocentric thalli. Despite this clear difference, commonly used genetic regions have never consistently separated the two genera as distinct lineages. The new study confirms that no single ribosomal marker region is sufficient to distinguish them, and that only when all ribosomal genetic regions are combined does a clear separation emerge.

To build the database, an international collaboration spanning Germany, the USA, New Zealand, India, China, the Czech Republic, the UK and Austria contributed pure cultures of anaerobic gut fungi from twelve research institutions. The collection included at least one isolate from each of the twenty genera formally described by the end of 2022, along with two newly discovered genus-level clades. Using Oxford Nanopore MinION and PacBio Sequel II sequencing, the team generated approximately 9 kilobase-pair operon sequences and developed a customized analysis pipeline called longread_umi that incorporates long-read pre-clustering and denoising steps to achieve high-confidence consensus sequences.

The resulting database comprises 770 consensus sequences covering the complete ribosomal operon and its adjacent intergenic regions. The mean length of a single operon copy was approximately 9 kilobase pairs. The researchers quantified sequence variability across every genetic region within the operon at four taxonomic levels: phylum, genus, species and strain. The intergenic spacer regions exhibited the highest variability, with IGS1 showing 84.6 percent sequence divergence across the phylum and IGS2 showing 72.5 percent. By contrast, the small subunit gene was extremely conserved, with only 0.48 percent variability across the entire phylum.

A significant correlation emerged between sequence variability and AT content throughout the operon. The functional genes ranged from 58.4 to 64.2 percent AT, while the non-coding transcribed spacer regions reached 71.7 to 81.8 percent AT. The non-transcribed intergenic spacers averaged 90 percent AT, consisting largely of repetitive motifs of approximately 25 base pairs. This compositional bias explains why the intergenic spacers are so variable: they are not under selective pressure to maintain a function linked to gene transcription.

The phylogenetic analysis incorporating both coding genes and non-coding regions, excluding the problematic IGS1, successfully resolved all examined genera into well-supported clades. The Cyllamyces and Caecomyces clades received 100 percent bootstrap support, and all seven analysed Piromyces species formed distinct species-level clades with full support. Even the Caecomyces varieties were distinguishable. The large ribosomal subunit gene provided the highest phylogenetic resolution among individual regions, while the external transcribed spacer region ETS1 proved unexpectedly valuable, providing an unambiguous resolution of Cyllamyces and Caecomyces that no other single marker could achieve.

Scanning the operon for markers suitable for short-read sequencing platforms revealed five potential regions. The most informative was Potential Marker 1, located at the end of ETS1 and the beginning of the small subunit gene. This approximately 230 base-pair region showed variability values closest to those of the established D1/D2 marker and resolved the Cyllamyces and Caecomyces clades, the Caecomyces varieties, and the seven Piromyces species. Its short length makes it suitable for Illumina sequencing and qPCR quantification, opening the door for culture-independent biodiversity surveys.

The researchers also documented fascinating structural features within the operon. The 5S ribosomal gene occurred in inverted orientations in 27.9 percent of all examined consensus sequences, a characteristic observed in seven of the twenty genera analysed. This inversion, previously documented in other eukaryotes including tilapia fish and various fungal genera, appears unrelated to the morphological characteristics of the fungi but could help distinguish Piromyces from other genera with similar traits. Both orientations might be transcribed, as TATA-like boxes were found downstream or at both ends of the gene.

The database represents the most comprehensive collection of ribosomal operon sequences from anaerobic gut fungi to date and is publicly available through GenBank and the Anaerobic Fungi Network website. By providing a reference backbone for phylogenetic analysis, it will accelerate the exploration of anaerobic gut fungal genomics, diversity, taxonomy and evolution. The identification of ETS1 and PM1 as alternative markers offers researchers powerful new tools for differentiating closely related lineages, while the quantified variability across every region of the operon provides a roadmap for designing future multi-marker studies that may finally settle whether Cyllamyces and Caecomyces are truly two genera or different morphotypes of one.

Subject of Research: Phylogenetic resolution of anaerobic gut fungi using full ribosomal operon sequencing

Article Title: Full ribosomal operon sequencing of anaerobic gut fungi (phylum Neocallimastigomycota ): insights on its markers and phylogenetic resolution

Article References: Young, D., Stüer-Patowsky, K., Huang, L., Elshahed, M. S., Youssef, N. H., Hanafy, R., Cheng, Y., Moon, C. D., Soni, P., Joshi, A., Stabel, M., Ochsenreither, K., Dagar, S. S., Hillman, E., Solomon, K. V., Fliegerová, K. O., Griffith, G. W., Callaghan, T. M., Podmirseg, S. M., … Wurzbacher, C. (2026). Full ribosomal operon sequencing of anaerobic gut fungi (phylum Neocallimastigomycota): insights on its markers and phylogenetic resolution. IMA Fungus, 17, Article e195921. https://doi.org/10.3897/imafungus.17.195921

Image Credits: AI Generated

DOI: 10.3897/imafungus.17.195921

Keywords: anaerobic gut fungi, Neocallimastigomycota, ribosomal operon, long-read sequencing, phylogenetics, ETS1, taxonomy, fungal evolution, Full, ribosomal, operon, sequencing