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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-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.

ABSTRACT The anaerobic gut fungi (AGF) inhabit the alimentary tracts of herbivores. In contrast to placental mammals, information regarding the identity, diversity, and community structure of AGF in marsupials is extremely sparse. Here, we characterized AGF communities in 61 fecal samples from 10 marsupial species belonging to four families in the order Diprotodontia: Vombatidae (wombats), Phascolarctidae (koalas), Phalangeridae (possums), and Macropodidae (kangaroos, wallabies, and pademelons). An amplicon-based diversity survey using the D2 region of the large ribosomal subunit as a phylogenetic marker indicated that marsupial AGF communities were dominated by eight genera commonly encountered in placental herbivores (Neocallimastix, Caecomyces, Cyllamyces, Anaeromyces, Orpinomyces, Piromyces, Pecoramyces, and Khoyollomyces). Community structure analysis revealed a high level of stochasticity, and ordination approaches did not reveal a significant role for the animal host, gut type, dietary preferences, or lifestyle in structuring marsupial AGF communities. Marsupial foregut and hindgut communities displayed diversity and community structure patterns comparable to AGF communities typically encountered in placental foregut hosts while exhibiting a higher level of diversity and a distinct community structure compared to placental hindgut communities. Quantification of AGF load using quantitative PCR indicated a significantly smaller load in marsupial hosts compared to their placental counterparts. Isolation efforts were only successful from a single red kangaroo fecal sample and yielded a Khoyollomyces ramosus isolate closely related to strains previously isolated from placental hosts. Our results suggest that AGF communities in marsupials are in low abundance and show little signs of selection based on ecological and evolutionary factors.IMPORTANCEThe 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.

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. IMPORTANCE The 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.

Nicrophorus is a genus of beetles that bury and transform small vertebrate carcasses into a brood ball coated with their oral and anal secretions to prevent decay and that will serve as a food source for their young. Nicrophorus pustulatus is an unusual species with the ability to overtake brood of other burying beetles and whose secretions, unlike other Nicrophorus species, has been reported not to exhibit antimicrobial properties. This work aims to better understand how the presence or absence of a food source influences the expression of genes involved in the feeding process of N. pustulatus. To achieve that, total RNA was extracted from pooled samples of salivary gland tissue from N. pustulatus and sequenced using an Illumina platform. The resulting reads were used to assemble a de novo transcriptome using Trinity. Duplicates with more than 95% similarity were removed to obtain a "unigene" set. Annotation of the unigene set was done using the Trinotate pipeline. Transcript abundance was determined using Kallisto and differential gene expression analysis was performed using edgeR. A total of 651 genes were found to be differentially expressed, including 390 upregulated and 261 downregulated genes in fed insects compared to starved. Several genes upregulated in fed beetles are associated with the insect immune response and detoxification processes with only one transcript encoding for the antimicrobial peptide (AMP) defensin. These results confirm that N. pustulatus does not upregulate the production of genes encoding AMPs during feeding. This study provides a snapshot of the changes in gene expression in the salivary glands of N. pustulatus following feeding while providing a well described transcriptome for the further analysis of this unique burying beetle.