Genetic profiling via a novel PCR-RFLP method enabled identification of four genera of anaerobic gut fungi isolated from nyala, giraffe, and zebra hosts

Herbivore gut microbiomes may contain a diversity of anaerobic gut fungi (AGF, phylum Neocallimastigomycota), important for fibre degradation. To perform functional studies and elucidate niches of AGF species, fungal isolates must be obtained into axenic culture, which is a resource-intensive process. Here we leverage the integration of morphological and functional assessments of AGF isolates with a newly developed PCR-RFLP strategy, to prioritise isolates of interest from faecal samples from zoo-housed animals for characterisation and confirmatory ITS or LSU sequencing. In silico prediction of PCR-RFLP profiles of cultured genera, followed by experimental validation, confirmed that LSU-based PCR-RFLP with AluI and Hyp188I digestion effectively identified fungi of distinct genera. Together our workflow resulted in isolation of a so far uncultured Piromyces (NY08) species and Neocallimastix cameroonii from nyala samples, as well as Feramyces austinii from giraffe and Khoyollomyces ramosus from zebra. Amplicon sequencing confirmed these species dominated AGF communities in their hosts, likely benefiting isolation success, and we identified enrichment conditions which also affected cultivability. The workflow developed here aids efficient AGF isolations, which are instrumental in expanding opportunities for functional studies that provide insight into the physiology and ecology of these fungi and help realise applications in white and green biotechnology.