Assessing anaerobic gut fungal diversity in herbivores using D1/D2 large ribosomal subunit sequencing and multi‐year isolation

Abstract

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 LSUbased 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.

Paucimyces polynucleatusgen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the feces of a wild blackbuck antelope

“…Similarly, the ITS1 region of strain BB-3 showed low intra strain sequence divergences (0-0.38%), and length (263-264 bp) heterogenicity. The ITS1 and D1/D2-LSU regions from strain BB-3 were 100% similar to sequences assigned to the uncultured lineage RH5 obtained in a previous culture-independent diversity survey from the same sample on which isolation was conducted (blackbuck deer), as well as few other samples (aoudad sheep, domesticated sheep, and Axis deer), demonstrating that these newly obtained isolates are cultured representatives of the RH5 lineage (Hanafy et al, 2020a).…”

“…A recent culture-independent survey of AGF diversity identified a wide range of novel yet-uncultured AGF lineages in fecal and rumen samples recovered from multiple herbivores in the states of Oklahoma and Texas (USA) (Hanafy et al, 2020a). Among the samples surveyed, a domesticated sheep and a wild blackbuck antelope showed a high relative abundance of the yet-uncultured lineage RH5 (96.2% and 52.4%, respectively) (Hanafy et al, 2020a). Due to the unavailability of sufficient feces from the domesticated sheep, isolation efforts were conducted only on the wild blackbuck antelope as previously described in (Hanafy et al, 2018; Stabel et al, 2020).…”

“…Nineteen anaerobic gut fungal (AGF) genera have been characterized so far (Barr et al, 1989; Breton et al, 1990; Callaghan et al, 2015; Dagar et al, 2015; Gold et al, 1988; Hanafy et al, 2017; Hanafy et al, 2018; Hanafy et al, 2020b; Heath et al, 1983; Joshi et al, 2018; Ozkose et al, 2001; Stabel et al, 2020). However, culture-independent diversity surveys have identified representatives of multiple yet-uncultured AGF genera (Hanafy et al, 2020a; Kittelmann et al, 2012; Liggenstoffer et al, 2010). The amenability of such lineages to isolation is uncertain.…”

“…Based on prior evidence (Hanafy et al, 2020a), we hypothesize that success in isolating a fungal taxon is directly proportional to its relative abundance within a specific sample. As such, targeting samples assessed to harbor a relatively large fraction of yet-uncultured taxa using culture-independent approaches should be prioritized in culture-based diversity efforts.…”

“…As such, targeting samples assessed to harbor a relatively large fraction of yet-uncultured taxa using culture-independent approaches should be prioritized in culture-based diversity efforts. During a recent culture-independent diversity survey of the AGF community in wild, zoo-housed, and domesticated herbivores in the US states of Oklahoma and Texas, we encountered several samples that harbored a high proportion of yet-uncultured genus-level clades of AGF (Hanafy et al, 2020a). We here report on the targeted isolation and detailed characterization of multiple strains belonging to one of these clades (lineage RH5).…”