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Abstract Anaerobic gut fungi (AGF,Neocallimastigomycota) represent a phylum of zoospore-producing fungi inhabiting the gastrointestinal tracts of herbivores. Twenty mammalian-affiliated genera (M-AGF) and two tortoise-affiliated genera (T-AGF) have been described so far. Here, we report on three additional novel T-AGF isolates obtained from Texas and sulcata tortoises. Phylogenetic analysis using the D1-D2 regions of the large ribosomal RNA subunit (D1-D2 LSU), RNA polymerase II large subunit (RPB1), internal transcribed spacer-1 region (ITS1), and transcriptomics-enabled phylogenomic analysis clustered these strains into three distinct, deep-branching clades, closely related to previously described T-AGF genusTestudinimyces. All isolates displayed filamentous rhizoidal growth patterns and produced monoflagellated zoospores. Unique morphological characteristics included the production of elongated, thick, nucleated structures in GX isolates, the formation of thin hair-like projections on sporangial walls in SR isolates, and irregularly shaped sporangia in TM isolates. All strains grew optimally at 32-35 °C and showed distinct substrate utilization capacity (e.g., growth on pectin, chitin, galactose). LSU analyses revealed GX isolates as the first cultured representatives of tortoise-affiliated but previously uncultured lineage NY56, while SR and TM strains have not been encountered in prior culture-independent AGF surveys. We propose to accommodate these isolates in three new genera and species –Gopheromyces tardescens(GXA2),Gigasporangiomyces pilosus(SR0.6), andKelyphomyces adhaerens(TM0.3). Further, based on the ecological, physiological, and phylogenetic distinctions between T-AGF and M-AGF, we propose to establish a new family (Testudinimycetaceae) to accommodate the generaTestudinimyces, Gopheromyces,Gigasporangiomyces,andKelyphomyces, within a new order (Testudinimycetales), and amend the description ofNeocallimastigalesto circumscribe M-AGF genera only. 

Abstract Anaerobic gut fungi (AGF; Neocallimastigomycota) are crucial for the degradation of plant biomass in herbivores. While extensively studied in mammals, information regarding their occurrence, diversity, and community structure in nonmammalian hosts remains sparse. Here, we report on the AGF community in fecal samples of 13 domesticated ostriches. The ostrich (Struthio camelus) is an herbivorous, flightless, hindgut-fermenting member of the class Aves (birds). Illumina-based metabarcoding targeting the D2 region of the large ribosomal subunit (28S rRNA) revealed a uniform AGF community with low alpha diversity in the fecal samples. The community was mostly comprised of sequences potentially representing two novel species in the genus Piromyces, and a novel genus in the Neocallimastigomycota. Sequences affiliated with these novel taxa were absent or extremely rare in datasets derived from mammalian and tortoise samples, indicating a strong pattern of AGF-host association. One Piromyces strain (strain Ost1) was successfully isolated. Transcriptomics-enabled molecular dating analysis suggested a divergence time of ≈ 30Mya, a time frame in line with current estimates for ostrich evolution. Comparative gene content analysis between strain Ost1 and other Piromyces species from mammalian sources revealed a high degree of similarity. Our findings expand the range of AGF animal hosts to include members of the birds (class Aves), highlight a unique AGF community in the ostrich alimentary tract, and document the occurrence of a strong pattern of fungal–host association in ostriches, similar to previously observed patterns in AGF canonical mammalian hosts. 

Abstract Anaerobic gut fungi (AGF, Neocallimastigomycota) inhabit the alimentary tract of herbivores. Although strict anaerobes, studies have suggested their capacity to retain viability after various durations of air exposure. It is currently unclear whether AGF can actively grow, and not merely survive, in redox potentials (Eh) higher than those encountered in the herbivorous gut. We evaluated the growth of two AGF strains (Orpinomyces joyonii and Testudinimyces gracilis) at various Eh levels, achieved by manipulating the concentrations of reductant (cysteine hydrochloride) in culture media. Both strains exhibited robust and sustainable growth at negative Eh (−50 mV or below). However, growth in the absence of cysteine hydrochloride (Eh value around +50 mV) was possible only for O. joyonii and only for one subcultivation. The capacity to grow at +50 mV was further confirmed in four additional taxa (Pecoramyces ruminatium, Anaeromyces mucronatus, Aklioshbmyces papillarum, and Piromyces communis), while two (Aestipascuomyces dupliciliberans and Capellomyces foraminis) failed to grow under these conditions. Our results establish the ability of AGF to grow at redox potential values higher than those encountered in their natural habitats. Such capability could contribute to efficient AGF dispersal and horizontal transmission between hosts, and could have important implications for industrial applications of AGF. 

ABSTRACT The rumen houses a diverse community that plays a major role in the digestion process in ruminants. Anaerobic gut fungi (AGF) are key contributors to plant digestion in the rumen. Here, we present a global amplicon-based survey of the rumen AGF mycobiome by examining 206 samples from 15 animal species, 15 countries, and 6 continents. The rumen AGF mycobiome was highly diverse, with 81 out of 88 currently recognized AGF genera or candidate genera identified. However, only six genera (Neocallimastix, Orpinomyces, Caecomyces, Cyllamyces,NY9, andPiromyces) were present at >4% relative abundance. AGF diversity was higher in members of the familiesAntilocapridaeandCervidaecompared toBovidae. Community structure analysis identified a pattern of phylosymbiosis, where host family (10% of total variance) and species (13.5%) partially explained the rumen mycobiome composition. As well, diet composition (9%–19%), domestication (11.14%), and biogeography (14.1%) also partially explained AGF community structure; although sampling limitation, geographic range restrictions, and direct association between different factors hindered accurate elucidation of the relative contribution of each factor. Pairwise comparison of rumen and fecal samples obtained from the same subject (n= 13) demonstrated greater diversity and inter-sample variability in rumen versus fecal samples. The generaNeocallimastixandOrpinomyceswere present in higher abundance in rumen samples, whileCyllamycesandCaecomyceswere enriched in fecal samples. Comparative analysis of global rumen and feces data sets revealed a similar pattern. Our results provide a global view of AGF community in the rumen and identify patterns of AGF variability between rumen and feces in herbivores Gastrointestinal (GI) tract. IMPORTANCERuminants are highly successful and economically important mammalian suborder. Ruminants are herbivores that digest plant material with the aid of microorganisms residing in their GI tract. In ruminants, the rumen compartment represents the most important location where microbially mediated plant digestion occurs, and is known to house a bewildering array of microbial diversity. An important component of the rumen microbiome is the anaerobic gut fungi (AGF), members of the phylumNeocallimastigomycota. So far, studies examining AGF diversity have mostly employed fecal samples, and little is currently known regarding the identity of AGF residing in the rumen compartment, factors that impact the observed patterns of diversity and community structure of AGF in the rumen, and how AGF communities in the rumen compare to AGF communities in feces. Here, we examined the rumen AGF diversity using an amplicon-based survey targeting a wide range of wild and domesticated ruminants (n= 206, 15 different animal species) obtained from 15 different countries. Our results demonstrate that while highly diverse, no new AGF genera were identified in the rumen mycobiome samples examined. Our analysis also indicate that animal host phylogeny, diet, biogeography, and domestication status could play a role in shaping AGF community structure. Finally, we demonstrate that a greater level of diversity and higher inter-sample variability was observed in rumen compared to fecal samples, with two genera (NeocallimastixandOrpinomyces) present in higher abundance in rumen samples, and two others (CyllamycesandCaecomyces) enriched in fecal samples. Our results provide a global view of the identity, diversity, and community structure of AGF in ruminants, elucidate factors impacting diversity and community structure of the rumen mycobiome, and identify patterns of AGF community variability between the rumen and feces in the herbivorous GI tract. 

Abstract 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-Neocallimastigomycotasymbiosis 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 inNeocallimastigomycota. 

Abstract Fungi are one of the most diverse groups of organisms with an estimated number of species in the range of 2–3 million. The higher-level ranking of fungi has been discussed in the framework of molecular phylogenetics since Hibbett et al., and the definition and the higher ranks (e.g., phyla) of the ‘true fungi’ have been revised in several subsequent publications. Rapid accumulation of novel genomic data and the advancements in phylogenetics now facilitate a robust and precise foundation for the higher-level classification within the kingdom. This study provides an updated classification of the kingdomFungi, drawing upon a comprehensive phylogenomic analysis ofHolomycota, with which we outline well-supported nodes of the fungal tree and explore more contentious groupings. We accept 19 phyla ofFungi,viz. Aphelidiomycota,Ascomycota,Basidiobolomycota,Basidiomycota,Blastocladiomycota,Calcarisporiellomycota,Chytridiomycota,Entomophthoromycota,Entorrhizomycota,Glomeromycota,Kickxellomycota,Monoblepharomycota,Mortierellomycota,Mucoromycota,Neocallimastigomycota,Olpidiomycota,Rozellomycota,Sanchytriomycota,andZoopagomycota. In the phylogenies,Caulochytriomycotaresides inChytridiomycota; thus, the former is regarded as a synonym of the latter, whileCaulochytriomycetesis viewed as a class inChytridiomycota. We provide a description of each phylum followed by its classes. A new subphylum,SanchytriomycotinaKarpov is introduced as the only subphylum inSanchytriomycota. The subclassPneumocystomycetidaeKirk et al. inPneumocystomycetes,Ascomycotais invalid and thus validated. Placements of fossil fungi in phyla and classes are also discussed, providing examples. 

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. 

Abstract Establishment of microbial communities in neonatal calves is vital for their growth and overall health. While this process has received considerable attention for bacteria, our knowledge on temporal progression of anaerobic gut fungi (AGF) in calves is lacking. Here, we examined AGF communities in faecal samples from six dairy cattle collected at 24 different time points during the pre‐weaning (days 1–48), weaning (days 48–60), and post‐weaning (days 60–360) phases. Quantitative polymerase chain reaction indicated that AGF colonisation occurs within 24 h after birth, with loads slowly increasing during pre‐weaning and weaning, then drastically increasing post‐weaning. Culture‐independent amplicon surveys identified higher alpha diversity during pre‐weaning/weaning, compared to post‐weaning. AGF community structure underwent a drastic shift post‐weaning, from a community enriched in genera commonly encountered in hindgut fermenters to one enriched in genera commonly encountered in adult ruminants.Comparison of AGF community between calves day 1 post‐birth and their mothers suggest a major role for maternal transmission, with additional input from cohabitating subjects. This distinct pattern of AGF progression could best be understood in‐light of their narrower niche preferences, metabolic specialisation, and physiological optima compared to bacteria, hence eliciting a unique response to changes in feeding pattern and associated structural GIT development during maturation.