Paucimyces polynucleatus gen. nov, sp. nov., a novel polycentric genus of anaerobic gut fungi from the faeces of a wild blackbuck antelope.

The anaerobic gut fungi (AGF; phylum Neocallimastigomycota) reside in the alimentary tracts of herbivores. Multiple novel, yet-uncultured AGF taxa have recently been identified in culture-independent diversity surveys. Here, we report on the isolation and characterization of the first representative of the RH5 lineage from faecal samples of a wild blackbuck (Indian Antelope, Antilope cervicapra) from Sutton County, Texas, USA. The isolates displayed medium sized (2-4 mm) compact circular colonies on agar roll tubes and thin loose biofilm-like growth in liquid medium. Microscopic examination revealed monoflagellated zoospores and polycentric thalli with highly branched nucleated filamentous rhizomycelium, a growth pattern encountered in a minority of described AGF genera so far. The obtained isolates are characterized by formation of spherical vesicles at the hyphal tips from which multiple sporangia formed either directly on the spherical vesicles or at the end of sporangiophores. Phylogenetic analysis using the D1/D2 regions of the large ribosomal subunit (D1/D2 LSU) and the ribosomal internal transcribed spacer 1 (ITS1) revealed sequence similarities of 93.5 and 81.3%, respectively, to the closest cultured relatives (Orpinomyces joyonii strain D3A (D1/D2 LSU) and Joblinomyces apicalis strain GFH681 (ITS1). Substrate utilization experiments using the type strain (BB-3T) demonstrated growth capabilities on a wide range of mono-, oligo- and polysaccharides, including glucose, xylose, mannose, fructose, cellobiose, sucrose, maltose, trehalose, lactose, cellulose, xylan, starch and raffinose. We propose accommodating these novel isolates in a new genus and species, for which the name Paucimyces polynucleatus gen. nov., sp. nov. is proposed.

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

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.

Phylogeny of anaerobic fungi (phylum Neocallimastigomycota), with contributions from yak in China.

The phylum Neocallimastigomycota contains eight genera (about 20 species) of strictly anaerobic fungi. The evolutionary relationships of these genera are uncertain due to insufficient sequence data to infer their phylogenies. Based on morphology and molecular phylogeny, thirteen isolates obtained from yak faeces and rumen digesta in China were assigned to Neocallimastix frontalis (nine isolates), Orpinomyces joyonii (two isolates) and Caecomyces sp. (two isolates), respectively. The phylogenetic relationships of the eight genera were evaluated using complete ITS and partial LSU sequences, compared to the ITS1 region which has been widely used in this phylum in the past. Five monophyletic lineages corresponding to six of the eight genera were statistically supported. Isolates of Caecomyces and Cyllamyces were present in a single lineage and could not be separated properly. Members of Neocallimastigomycota with uniflagellate zoospores represented by Piromyces were polyphyletic. The Piromyces-like genus Oontomyces was consistently closely related to the traditional Anaeromyces, and separated the latter genus into two clades. The phylogenetic position of the Piromyces-like genus Buwchfawromyces remained unresolved. Orpinomyces and Neocallimastix, sharing polyflagellate zoospores, were supported as sister genera in the LSU phylogeny. Apparently ITS, specifically ITS1 alone, is not a good marker to resolve the generic affinities of the studied fungi. The LSU sequences are easier to align and appear to work well to resolve generic relationships. This study provides a comparative phylogenetic revision of Neocallimastigomycota isolates known from culture and sequence data.

Finding a robust strain for biomethanation: anaerobic fungi (Neocallimastigomycota) from the Alpine ibex (Capra ibex) and their associated methanogens.

Anaerobic fungi occupy the rumen and digestive tract of herbivores, where they play an important role in enzymatic digestion of lignocellulosic and cellulosic substrates, i.e. organic material that their hosts are unable to decompose on their own. In this study we isolated anaerobic fungi from a typical alpine herbivore, the Alpine ibex (C. ibex). Three fungal strains, either as pure culture (ST2) or syntrophic co-culture with methanogens (ST3, ST4) were successfully obtained and morphologically characterised by different microscopy- and staining-techniques and by rDNA ITS gene sequencing. The isolated fungi were identified as Neocallimastix frontalis (ST2) and Caecomyces communis (ST3 and ST4). We introduce a novel field of application for lactofuchsin-staining, combined with confocal laser scanning microscopy. This approach proved as an effective method to visualize fungal structures, especially in the presence of plant biomass, generally exhibiting high autofluorescence. Moreover, we could demonstrate that fungal morphology is subject to changes depending on the carbon source used for cultivation. Oxygen tolerance was confirmed for both, C. communis-cultures for up to three, and for the N. frontalis-isolate for up to 12 h, respectively. With PCR, FISH and an oligonucleotide microarray we found associated methanogens (mainly Methanobacteriales) for C. communis, but not for N. frontalis.

Seven new Neocallimastigomycota genera from wild, zoo-housed, and domesticated herbivores greatly expand the taxonomic diversity of the phylum.

We isolated and characterized 65 anaerobic gut fungal (AGF; Neocallimastigomycota) strains from fecal samples of five wild (W, axis deer, white-tailed deer, Boer goat, mouflon, and Nilgiri tahr), one zoo-housed (Z, zebra), and three domesticated (D,  horse, sheep, and goat) herbivores in the US states of Texas (TX) and Oklahoma (OK), Wales (WA), and the Indian states of Kerala (KE) and Haryana (HA). Phylogenetic assessment using the D1-D2 regions of the large subunit (28S) rDNA and internal transcribed spacer 1 (ITS1) identified seven monophyletic clades that are distinct from all currently recognized AGF genera. All strains displayed monocentric thalli and produced exclusively or predominantly monoflagellate zoospores, with the exception of axis deer strains, which produced polyflagellate zoospores. Analysis of amplicon-based AGF diversity surveys indicated that zebra and horse strains are representatives of uncultured AL1 group, whereas domesticated goat and sheep strains are representatives of uncultured AL5 group, previously encountered in fecal and rumen samples of multiple herbivores. The other five lineages, all of which were isolated from wild herbivores, have not been previously encountered in such surveys. Our results significantly expand the genus-level diversity within the Neocallimastigomycota and strongly suggest that wild herbivores represent a yet-untapped reservoir of AGF diversity. We propose seven novel genera and eight novel Neocallimastigomycota species to comprise these strains, for which we propose the names Agriosomyces longus (mouflon and wild Boer goat), Aklioshbomyces papillarum (white-tailed deer), Capellomyces foraminis (wild Boar goat), and C. elongatus (domesticated goat), Ghazallomyces constrictus (axis deer), Joblinomyces apicalis (domesticated goat and sheep), Khoyollomyces ramosus (zebra-horse), and Tahromyces munnarensis (Nilgiri tahr).

A fast and reliable procedure for spore collection from anaerobic fungi: Application for RNA uptake and long-term storage of isolates.

Anaerobic gut fungi (AGF) represent a basal fungal lineage (phylum Neocallimastigomycota) that resides in the rumen and alimentary tracts of herbivores. The AGF reproduce asexually, with a life cycle that involves flagellated zoospores released from zoosporangia followed by encystment, germination and the subsequent development of rhizomycelia. A fast and reliable approach for AGF spore collection is critical not only for developmental biology studies, but also for molecular biological (e.g. AMT-transformation and RNAi) approaches. Here, we developed and optimized a simple and reliable procedure for the collection of viable, competent, and developmentally synchronized AGF spores under strict anaerobic conditions. The approach involves growing AGF on agar medium in serum bottles under anaerobic conditions, and flooding the observed aerial growth to promote spore release from sporangia into the flooding suspension. The released spores are gently collected using a wide bore sterile needle. Process optimization resulted in the recovery of up to 7×10(9) spores per serum bottle. Further, the released spores exhibited synchronized development from flagellated spores to encysted spores and finally to germinating spores within 90min from the onset of flooding. At the germinating spore stage, the obtained spores were competent, and readily uptook small interfering RNA (siRNA) oligonucleotides. Finally, using multiple monocentric and polycentric AGF isolates, we demonstrate that AGF grown on agar surface could retain viability for up to 16weeks at 39°C, and hence this solid surface growth procedure represents a simple, cryopreservative- and freezing temperature-free approach for AGF storage.

Development of three specific PCR-based tools to determine quantity, cellulolytic transcriptional activity and phylogeny of anaerobic fungi.

Anaerobic fungi (AF) decompose plant material with their rhizoid and multiple cellulolytic enzymes. They disintegrate the complex structure of lignocellulosic substrates, making them more accessible and suitable for further microbial degradation. There is also much interest in their use as biocatalysts for biotechnological applications. Here, three novel polymerase chain reaction (PCR)-based methods for detecting AF and their transcriptional activity in in vitro cultures and environmental samples were developed. Two real-time quantitative PCR (qPCR)-based methods targeting AF were developed: AF-SSU, was designed to quantify the 18S rRNA genes of AF. AF-Endo, measuring transcripts of an endoglucanase gene from the glycoside hydrolase family 5 (GH5), was developed to quantify their transcriptional cellulolytic activity. The third PCR based approach was designed for phylogenetical analysis. It targets the 28S rRNA gene (LSU) of AF revealing their phylogenetic affiliation. The in silico-designed primer/probe combinations were successfully tested for the specific amplification of AF from animal and biogas plant derived samples. In combination, these three methods represent useful tools for the analysis of AF transcriptional cellulolytic activity, their abundance and their phylogenetic placement.

Phylogenetic diversity and community structure of anaerobic gut fungi (phylum Neocallimastigomycota) in ruminant and non-ruminant herbivores.

The phylogenetic diversity and community structure of members of the gut anaerobic fungi (AF) (phylum Neocallimastigomycota) were investigated in 30 different herbivore species that belong to 10 different mammalian and reptilian families using the internal transcribed spacer region-1 (ITS-1) ribosomal RNA (rRNA) region as a phylogenetic marker. A total of 267 287 sequences representing all known anaerobic fungal genera were obtained in this study. Sequences affiliated with the genus Piromyces were the most abundant, being encountered in 28 different samples, and representing 36% of the sequences obtained. On the other hand, sequences affiliated with the genera Cyllamyces and Orpinomyces were the least abundant, being encountered in 2, and 8 samples, and representing 0.7%, and 1.1% of the total sequences obtained, respectively. Further, 38.3% of the sequences obtained did not cluster with previously identified genera and formed eight phylogenetically distinct novel anaerobic fungal lineages. Some of these novel lineages were widely distributed (for example NG1 and NG3), whereas others were animal specific, being encountered in only one or two animals (for example NG4, NG6, NG7, and NG8). The impact of various physiological and environmental factors on the diversity and community structure of AF was examined. The results suggest that animal host phylogeny exerts the most significant role on shaping anaerobic fungal diversity and community composition. These results greatly expand the documented global phylogenetic diversity of members of this poorly studied group of fungi that has an important function in initiating plant fiber degradation during fermentative digestion in ruminant and non-ruminant herbivores.