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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). 

Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear criteria for taxa characterization and rank assignment. Within the anaerobic fungi (phylum Neocallimastigomycota), the need for such criteria is especially vital. Difficulties associated with their isolation, maintenance and long-term storage often result in limited availability and loss of previously described taxa. To this end, we provide here a list of morphological, microscopic, phylogenetic and phenotypic criteria for assessment and documentation when characterizing newly obtained Neocallimastigomycota isolates. We also recommend a polyphasic rank-assignment scheme for novel genus-, species- and strain-level designations for newly obtained Neocallimastigomycota isolates. 

Abstract Despite their role in host nutrition, the anaerobic gut fungal (AGF) component of the herbivorous gut microbiome remains poorly characterized. Here, to examine global patterns and determinants of AGF diversity, we generate and analyze an amplicon dataset from 661 fecal samples from 34 mammalian species, 9 families, and 6 continents. We identify 56 novel genera, greatly expanding AGF diversity beyond current estimates (31 genera and candidate genera). Community structure analysis indicates that host phylogenetic affiliation, not domestication status and biogeography, shapes the community rather than. Fungal-host associations are stronger and more specific in hindgut fermenters than in foregut fermenters. Transcriptomics-enabled phylogenomic and molecular clock analyses of 52 strains from 14 genera indicate that most genera with preferences for hindgut hosts evolved earlier (44-58 Mya) than those with preferences for foregut hosts (22-32 Mya). Our results greatly expand the documented scope of AGF diversity and provide an ecologically and evolutionary-grounded model to explain the observed patterns of AGF diversity in extant animal hosts.