Anaerobic fungi contain abundant, diverse, and transcriptionally active Long Terminal Repeat retrotransposons.

Long Terminal Repeat (LTR) retrotransposons are a class of repetitive elements that are widespread in the genomes of plants and many fungi. LTR retrotransposons have been associated with rapidly evolving gene clusters in plants and virulence factor transfer in fungal-plant parasite-host interactions. We report here the abundance and transcriptional activity of LTR retrotransposons across several species of the early-branching Neocallimastigomycota, otherwise known as the anaerobic gut fungi (AGF). The ubiquity of LTR retrotransposons in these genomes suggests key evolutionary roles in these rumen-dwelling biomass degraders, whose genomes also contain many enzymes that are horizontally transferred from other rumen-dwelling prokaryotes. Up to 10% of anaerobic fungal genomes consist of LTR retrotransposons, and the mapping of sequences from LTR retrotransposons to transcriptomes shows that the majority of clusters are transcribed, with some exhibiting expression greater than 104 reads per kilobase million mapped reads (rpkm). Many LTR retrotransposons are strongly differentially expressed upon heat stress during fungal cultivation, with several exhibiting a nearly three-log10 fold increase in expression, whereas growth substrate variation modulated transcription to a lesser extent. We show that some LTR retrotransposons contain carbohydrate-active enzymes (CAZymes), and the expansion of CAZymes within genomes and among anaerobic fungal species may be linked to retrotransposon activity. We further discuss how these widespread sequences may be a source of promoters and other parts towards the bioengineering of anaerobic fungi.

Anaerobic fungi in the tortoise alimentary tract illuminate early stages of host-fungal symbiosis and Neocallimastigomycota evolution.

Anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tract of herbivores. While their presence in mammals is well documented, evidence for their occurrence in non-mammalian hosts is currently sparse. Culture-independent surveys of AGF in tortoises identified a unique community, with three novel deep-branching genera representing >90% of sequences in most samples. Representatives of all genera were successfully isolated under strict anaerobic conditions. Transcriptomics-enabled phylogenomic and molecular dating analyses indicated an ancient, deep-branching position in the AGF tree for these genera, with an evolutionary divergence time estimate of 104-112 million years ago (Mya). Such estimates push the establishment of animal-Neocallimastigomycota symbiosis from the late to the early Cretaceous. Further, tortoise-associated isolates (T-AGF) exhibited limited capacity for plant polysaccharides metabolism and lacked genes encoding several carbohydrate-active enzyme (CAZyme) families. Finally, we demonstrate that the observed curtailed degradation capacities and reduced CAZyme repertoire is driven by the paucity of horizontal gene transfer (HGT) in T-AGF genomes, compared to their mammalian counterparts. This reduced capacity was reflected in an altered cellulosomal production capacity in T-AGF. Our findings provide insights into the phylogenetic diversity, ecological distribution, evolutionary history, evolution of fungal-host nutritional symbiosis, and dynamics of genes acquisition in Neocallimastigomycota.

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.

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).

Horizontal Gene Transfer as an Indispensable Driver for Evolution of Neocallimastigomycota into a Distinct Gut-Dwelling Fungal Lineage.

Survival and growth of the anaerobic gut fungi (AGF; Neocallimastigomycota) in the herbivorous gut necessitate the possession of multiple abilities absent in other fungal lineages. We hypothesized that horizontal gene transfer (HGT) was instrumental in forging the evolution of AGF into a phylogenetically distinct gut-dwelling fungal lineage. The patterns of HGT were evaluated in the transcriptomes of 27 AGF strains, 22 of which were isolated and sequenced in this study, and 4 AGF genomes broadly covering the breadth of AGF diversity. We identified 277 distinct incidents of HGT in AGF transcriptomes, with subsequent gene duplication resulting in an HGT frequency of 2 to 3.5% in AGF genomes. The majority of HGT events were AGF specific (91.7%) and wide (70.8%), indicating their occurrence at early stages of AGF evolution. The acquired genes allowed AGF to expand their substrate utilization range, provided new venues for electron disposal, augmented their biosynthetic capabilities, and facilitated their adaptation to anaerobiosis. The majority of donors were anaerobic fermentative bacteria prevalent in the herbivorous gut. This study strongly indicates that HGT indispensably forged the evolution of AGF as a distinct fungal phylum and provides a unique example of the role of HGT in shaping the evolution of a high-rank taxonomic eukaryotic lineage.IMPORTANCE The anaerobic gut fungi (AGF) represent a distinct basal phylum lineage (Neocallimastigomycota) commonly encountered in the rumen and alimentary tracts of herbivores. Survival and growth of anaerobic gut fungi in these anaerobic, eutrophic, and prokaryote-dominated habitats necessitates the acquisition of several traits absent in other fungal lineages. We assess here the role of horizontal gene transfer as a relatively fast mechanism for trait acquisition by the Neocallimastigomycota postsequestration in the herbivorous gut. Analysis of 27 transcriptomes that represent the broad diversity of Neocallimastigomycota identified 277 distinct HGT events, with subsequent gene duplication resulting in an HGT frequency of 2 to 3.5% in AGF genomes. These HGT events have allowed AGF to survive in the herbivorous gut by expanding their substrate utilization range, augmenting their biosynthetic pathway, providing new routes for electron disposal by expanding fermentative capacities, and facilitating their adaptation to anaerobiosis. HGT in the AGF is also shown to be mainly a cross-kingdom affair, with the majority of donors belonging to the bacteria. This study represents a unique example of the role of HGT in shaping the evolution of a high-rank taxonomic eukaryotic lineage.

The biotechnological potential of anaerobic fungi on fiber degradation and methane production.

Anaerobic fungi (phylum Neocallimastigomycota), an early branching family of fungi, are commonly encountered in the digestive tract of mammalian herbivores. To date, isolates from ten described genera have been reported, and several novel taxonomic groupings are detected using culture-independent molecular methods. Anaerobic fungi are recognized as playing key roles in the decomposition of lignocellulose (up to 50% of the ingested and untreated lignocellulose), with their physical penetration and extracellular enzymatical secretion of an unbiased diverse repertoire of cell-wall-degrading enzymes. The secreted cell-wall-degrading enzymes of anaerobic fungi include both free enzymes and extracellular multi-enzyme complexes called cellulosomes, both of which have potential as fiber degraders in industries. In addition, anaerobic fungi can provide large amounts of substrates such as hydrogen, formate, and acetate for their co-cultured methanogens. Consequently, large amounts of methane can be produced. And thus, it is promising to use the co-culture of anaerobic fungi and methanogens in the biogas process to intensify the biogas yield owing to the efficient and robust degradation of recalcitrant biomass by anaerobic fungi and improved methane production from co-cultures of anaerobic fungi and methanogens.

Catabolic repression in early-diverging anaerobic fungi is partially mediated by natural antisense transcripts.

Early-diverging anaerobic fungi (order: Neocallimastigomycota), lignocelluolytic chytrid-like fungi central to fiber degradation in the digestive tracts of large herbivores, are attractive sources of cellulases and hemicellulases for biotechnology. Enzyme expression is tightly regulated and coordinated through mechanisms that remain unelucidated to optimize hydrolytic efficiency. Our analysis of anaerobic fungal transcriptomes reveals hundreds of cis-natural antisense transcripts (cis-NATs), which we hypothesize play an integral role in this regulation. Through integrated genomic and transcriptomic sequencing on a range of catabolic substrates, we validate these NATs in three species (Anaeromyces robustus, Neocallimasix californiae, and Piromyces finnis), and analyze their expression patterns and prevalence to gain insight into their function. NAT function was diverse and conserved across the three fungal genomes studied, with 10% of all metabolic process NATs associated with lignocellulose hydrolysis. Despite these similarities, however, only eleven gene targets were conserved orthologs. Several NATs were dynamically regulated by lignocellulosic substrates while their gene targets were unregulated. This observation is consistent with a hypothesized, but untested, regulatory mechanism where selected genes are exclusively regulated at the transcriptional/post-transcriptional level by NATs. However, only genes with high NAT relative expression levels displayed this phenomenon, suggesting a selection mechanism that favors larger dynamic ranges for more precise control of gene expression. In addition to this mode, we observed two other possible regulatory fates: canonical transcriptional regulation with no NAT response, and positive co-regulation of target mRNA and cognate NAT, which we hypothesize is a fine-tuning strategy to locally negate control outputs from global regulators. Our work reveals the complex contributions of antisense RNA to the catabolic response in anaerobic fungi, highlighting its importance in understanding lignocellulolytic activity for bioenergy applications. More importantly, the relative expression of NAT to target may form a critical determinant of transcriptional vs post-transcriptional (NAT) control of gene expression in primitive anaerobic fungi.

Methods for Genomic Characterization and Maintenance of Anaerobic Fungi.

The rapid development of molecular biology and bioinformatics has fueled renewed interests in anaerobic fungi from the phylum Neocallimastigomycota. This chapter presents well-established methods for isolation, routine cultivation, and cryopreservation of anaerobic fungi. Moreover, detailed nucleic acid extraction protocols are provided, which should enable readers to isolate high-quality DNA and RNA from a variety of anaerobic fungal culture media for downstream applications such as next-generation sequencing.

Pecoramyces ruminantium, gen. nov., sp. nov., an anaerobic gut fungus from the feces of cattle and sheep.

The anaerobic gut fungi (AGF) inhabit the rumen and alimentary tracts of multiple ruminant and nonruminant herbivores, belong to a distinct phylum-level lineage (Neocallimastigomycota), and play an important role in plant biomass degradation in many herbivores. As part of a wider effort to obtain AGF with high lignocellulolytic capacities, we isolated and characterized four different AGF strains from the feces of cattle and sheep. Microscopically, isolates produced monocentric thalli and monoflagellated zoospores. Phylogenetic analysis revealed that all isolates formed a monophyletic cluster with strong bootstrap support as a sister clade to the genus Orpinomyces and close to Neocallimastix, an unexpected result because these two genera of AGF form polyflagellated zoospores. Isolates displayed a smooth biofilm-like growth in liquid medium and formed small (0.5-1 mm) pinpoint circular colonies on agar roll tubes. Both endogenous and exogenous sporangia were observed with variable shapes and sizes. Zoospores were mainly spherical, with diameters ranging between 3.8 and 12.5 µm, and mostly a single flagellum. All strains exhibited similar substrate utilization patterns and comparable cellulolytic and xylanolytic activities. Similar ITS1 sequences falling within the same distinctive clade were found on GenBank, with all environmental samples obtained from diverse ruminant and pseudoruminant hosts from three continents, but not from any hindgut-fermenting hosts. Given the high level of sequence divergence between our strains and closest cultured representatives and their distinct microscopic/macroscopic features, we propose a new genus, Pecoramyces, from the name of the taxonomic infraorder Pecora ("horned ruminants" or "higher ruminants"; derived from the Latin word for horned livestock), and a new species, P. ruminantium (since occurrence seems to be specific to ruminant/pseudoruminant foregut, but not hindgut-fermenting mammals).

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.

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.

Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota).

The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

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.

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.