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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 In the central plains of North America, the beetle family Silphidae comprised two subfamilies, Silphinae and Nicrophorinae, differentiated by reproductive behaviors. Silphinae, known as carrion beetles, feed on carrion and fly larvae and produce free-living larvae that receive no parental care. Adult Nicrophorinae, known as burying beetles, prepare a vertebrate carcass into a brood ball and provide biparental care to their offspring. Preparation of a brood ball involves coating the carcass in antimicrobial oral and anal secretions. These secretions contain a community of microbes, referred to as the secretion microbiome, which inhibit carcass microbe succession, preventing normal decomposition. Here, the secretion microbiomes of five species of Nicrophorinae and two species of Silphinae, both sampled from Oklahoma, with additional Nicrophorinae from Nebraska, were characterized using culture-independent analyses to understand and decipher factors shaping diversity and community structure. We identify the core secretion microbiome across Silphidae and show that, while the host subfamily, secretion type, and collection locality had no significant effect on the bacterial community alpha diversity, these factors significantly influenced bacterial community structure. Global and local tests of phylogenetic associations identified 14 genera with phylogenetic signals to the host subfamily and species. Description of the bacterial communities present in silphid secretions furthers our understanding of how these beetles interact with microbes for carcass nutrient processing. Future culture-dependent studies from silphid secretions may identify novel antimicrobials and nontoxic compounds that can act as meat preservatives or sources for antimicrobials. IMPORTANCEThe manuscript explores the secretion bacterial community of carrion and burying beetles of the central plains of North America. A core secretion microbiome of 11 genera is identified. The host subfamily, secretion type, and collection locality significantly affects the secretion microbiome. Future culture-dependent studies from silphid secretions may identify novel antimicrobials and nontoxic compounds that can act as meat preservatives or sources for antimicrobials. 

We report on the isolation and characterization of three isolates of anaerobic gut fungi from a cattle faecal sample obtained in Stillwater, OK, USA. The isolates produced polycentric thalli with nucleated rhizomycelia, lobed appressorium-like structures, intercalary sporangia and constricted sausage-like hyphae. These morphological features are characteristic of members of the genusAnaeromyces. No zoospore production was observed during the isolation process or thereafter. The strains seemed to have propagated solely through their nucleated hyphae post initial enrichment. Phylogenetic analysis of the D1/D2 region of the large ribosomal subunit (D1/D2 LSU) rRNA, the ribosomal intergenic spacer region 1 (ITS1), RNA polymerase II large subunit (RPB1) and comparative average amino acid identity using transcriptomic datasets further confirmed the position of the type strain as a distinct member of the genusAnaeromyces, familyAnaeromycetaceaeand phylumNeocallimastigomycota. We propose to accommodate these isolates into a new species (Anaeromyces corallioides) within the genusAnaeromyces. The type strain is EE.1. 

The anaerobic gut fungi (AGF,Neocallimastigomycota) represent a basal zoosporic phylum within the kingdomFungi. Twenty genera are currently described, all of which were isolated from the digestive tracts of mammalian herbivores. Here, we report on the isolation and characterization of novel AGF taxa from faecal samples of tortoises. Twenty-nine fungal isolates were obtained from seven different tortoise species. Phylogenetic analysis using the D1/D2 region of the LSU rRNA gene, ribosomal internal transcribed spacer 1, and RNA polymerase II large subunit grouped all isolates into two distinct, deep-branching clades (clades T and B), with a high level of sequence divergence to their closest cultured relative (Khoyollomyces ramosus). Average amino acid identity values calculated using predicted peptides from the isolates’ transcriptomes ranged between 60.80–66.21  % (clade T), and 61.24–64.83  % (clade B) when compared to all other AGF taxa; values that are significantly below recently recommended thresholds for genus (85%) and family (75%) delineation in theNeocallimastigomycota. Both clades displayed a broader temperature growth range (20–45 °C, optimal 30 °C for clade T, and 30–42 °C, optimal 39 °C for clade B) compared to all other AGF taxa. Microscopic analysis demonstrated that strains from both clades produced filamentous hyphae, polycentric rhizoidal growth patterns, and monoflagellated zoospores. Isolates in clade T were characterized by the production of unbranched, predominantly narrow hyphae, and small zoospores, while isolates in clade B were characterized by the production of multiple sporangiophores and sporangia originating from a single central swelling resulting in large multi-sporangiated structures. Based on the unique phylogenetic positions, AAI values, and phenotypic characteristics, we propose to accommodate these isolates into two novel genera (TestudinimycesandAstrotestudinimyces), and species (T. gracilisandA. divisus) within the orderNeocallimastigales. The type species are strains T130A^T(T. gracilis) and B1.1^T(A. divisus). 

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. 

Members of the anaerobic gut fungi ( Neocallimastigomycota ) reside in the rumen and alimentary tract of larger mammalian and some reptilian, marsupial and avian herbivores. The recent decade has witnessed a significant expansion in the number of described Neocallimastigomycota genera and species. However, the difficulties associated with the isolation and maintenance of Neocallimastigomycota strains has greatly complicated comparative studies to resolve inter- and intra-genus relationships. Here, we provide an updated outline of Neocallimastigomycota taxonomy. We critically evaluate various morphological, microscopic and phylogenetic traits previously and currently utilized in Neocallimastigomycota taxonomy, and provide an updated key for quick characterization of all genera. We then synthesize data from taxa description manuscripts, prior comparative efforts and molecular sequence data to present an updated list of Neocallimastigomycota genera and species, with an emphasis on resolving relationships and identifying synonymy between recent and historic strains. We supplement data from published manuscripts with information and illustrations from strains in the authors’ collections. Twenty genera and 36 species are recognized, but the status of 10 species in the genera Caecomyces, Piromyces , Anaeromyces and Cyllamyces remains uncertain due to the unavailability of culture and conferre ( cf .) strains, lack of sequence data, and/or inadequacy of available microscopic and phenotypic data. Six cases of synonymy are identified in the genera Neocallimastix and Caecomyces , and two names in the genus Piromyces are rejected based on apparent misclassification. 

The AGF are integral part of the microbiome of herbivores. They play a crucial role in breaking down plant biomass in hindgut and foregut fermenters. The majority of research has been conducted on the AGF community in placental mammalian hosts. However, it is important to note that many marsupial mammals are also herbivores and employ a hindgut or foregut fermentation strategy for breaking down plant biomass. So far, very little is known regarding the AGF diversity and community structure in marsupial mammals. To fill this knowledge gap, we conducted an amplicon-based diversity survey targeting AGF in 61 fecal samples from 10 marsupial species. We hypothesize that, given the distinct evolutionary history and alimentary tract architecture, novel and unique AGF communities would be encountered in marsupials. Our results indicate that marsupial AGF communities are highly stochastic, present in relatively low loads, and display community structure patterns comparable to AGF communities typically encountered in placental foregut hosts. Our results indicate that marsupial hosts harbor AGF communities; however, in contrast to the strong pattern of phylosymbiosis typically observed between AGF and placental herbivores, the identity and gut architecture appear to play a minor role in structuring AGF communities in marsupials. 

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.