The anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tracts of herbivores where they play a central role in the breakdown of plant material. Here, we report on the development of the hypervariable domains D1/D2 of the large ribosomal subunit (D1/D2 LSU) as a barcoding marker for the AGF. We generated a reference D1/D2 LSU database for all cultured AGF genera, as well as the majority of candidate genera encountered in prior internal transcribed spacer 1 (ITS1)‐based surveys. Subsequently, a D1/D2 LSU‐based diversity survey using long read PacBio SMRT sequencing was conducted on faecal samples from 21 wild and domesticated herbivores. Twenty‐eight genera and candidate genera were identified, including multiple novel lineages that were predominantly, but not exclusively, identified in wild herbivores. Association between certain AGF genera and animal lifestyles, or animal host family was observed. Finally, to address the current paucity of AGF isolates, concurrent isolation efforts utilizing multiple approaches to maximize recovery yielded 216 isolates belonging to 12 different genera, several of which have no prior cultured‐representatives. Our results establish the utility of D1/D2 LSU and PacBio sequencing for AGF diversity surveys, the culturability of multiple AGF taxa, and demonstrate that wild herbivores represent a yet‐untapped reservoir of AGF diversity.
- Award ID(s):
- 2029478
- NSF-PAR ID:
- 10299057
- Date Published:
- Journal Name:
- International Journal of Systematic and Evolutionary Microbiology
- Volume:
- 71
- Issue:
- 6
- ISSN:
- 1466-5026
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary -
The anaerobic gut fungi (AGF,
Neocallimastigomycota ) represent a basal zoosporic phylum within the kingdomFungi . Twenty genera are currently described, all of which were isolated from the digestive tracts of mammalian herbivores. Here, we report on the isolation and characterization of novel AGF taxa from faecal samples of tortoises. Twenty-nine fungal isolates were obtained from seven different tortoise species. Phylogenetic analysis using the D1/D2 region of the LSU rRNA gene, ribosomal internal transcribed spacer 1, and RNA polymerase II large subunit grouped all isolates into two distinct, deep-branching clades (clades T and B), with a high level of sequence divergence to their closest cultured relative (Khoyollomyces ramosus ). Average amino acid identity values calculated using predicted peptides from the isolates’ transcriptomes ranged between 60.80–66.21 % (clade T), and 61.24–64.83 % (clade B) when compared to all other AGF taxa; values that are significantly below recently recommended thresholds for genus (85%) and family (75%) delineation in theNeocallimastigomycota . Both clades displayed a broader temperature growth range (20–45 °C, optimal 30 °C for clade T, and 30–42 °C, optimal 39 °C for clade B) compared to all other AGF taxa. Microscopic analysis demonstrated that strains from both clades produced filamentous hyphae, polycentric rhizoidal growth patterns, and monoflagellated zoospores. Isolates in clade T were characterized by the production of unbranched, predominantly narrow hyphae, and small zoospores, while isolates in clade B were characterized by the production of multiple sporangiophores and sporangia originating from a single central swelling resulting in large multi-sporangiated structures. Based on the unique phylogenetic positions, AAI values, and phenotypic characteristics, we propose to accommodate these isolates into two novel genera (Testudinimyces andAstrotestudinimyces ), and species (T. gracilis andA. divisus ) within the orderNeocallimastigales . The type species are strains T130AT(T. gracilis ) and B1.1T(A. divisus ). -
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