Diversity and community structure of anaerobic gut fungi in the rumen of wild and domesticated herbivores.

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, and Piromyces) were present at >4% relative abundance. AGF diversity was higher in members of the families Antilocapridae and Cervidae compared to Bovidae. 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 genera Neocallimastix and Orpinomyces were present in higher abundance in rumen samples, while Cyllamyces and Caecomyces were 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 phylum Neocallimastigomycota. 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 (Neocallimastix and Orpinomyces) present in higher abundance in rumen samples, and two others (Cyllamyces and Caecomyces) 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.

Effect of dietary macronutrients and immune challenge on gut microbiota, physiology and feeding behaviour in zebra finches.

Macronutrients play a vital role in host immunity and can influence host-pathogen dynamics, potentially through dietary effects on gut microbiota. To increase our understanding of how dietary macronutrients affect physiology and gut microbiota and investigate whether feeding behaviour is influenced by an immune threat, we conducted two experiments. First, we determined whether zebra finches (Taeniopygia guttata) exhibit shifts in physiology and gut microbiota when fed diets differing in macronutrient ratios. We found the type and amount of diet consumed affected gut microbiota alpha diversity, where microbial richness and Shannon diversity increased with caloric intake in birds fed a high-fat diet and decreased with caloric intake in birds fed a high protein diet. Diet macronutrient content did not affect physiological metrics, but lower caloric intake was associated with higher complement activity. In our second experiment, we simulated an infection in birds using the bacterial endotoxin lipopolysaccharide (LPS) and quantified feeding behaviour in immune challenged and control individuals, as well as birds housed near either a control pair (no immune threat), or birds housed near a pair given an immune challenge with LPS (social cue of heightened infection risk). We also examined whether social cues of infection alter physiological responses relevant to responding to an immune threat, an effect that could be mediated through shifts in feeding behaviour. LPS induced a reduction in caloric intake driven by a decrease in protein, but not fat consumption. No evidence was found for socially induced shifts in feeding behaviour, physiology or gut microbiota. Our findings carry implications for host health, as sickness-induced anorexia and diet-induced shifts in the microbiome could shape host-pathogen interactions.

Temporal progression of anaerobic fungal communities in dairy calves from birth to maturity.

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.

Physiological and genomic insights into abiotic stress of halophilic archaeon Natrinema altunense 4.1R isolated from a saline ecosystem of Tunisian desert.

Halophilic archaea are polyextremophiles with the ability to withstand fluctuations in salinity, high levels of ultraviolet radiation, and oxidative stress, allowing them to survive in a wide range of environments and making them an excellent model for astrobiological research. Natrinema altunense 4.1R is a halophilic archaeon isolated from the endorheic saline lake systems, Sebkhas, located in arid and semi-arid regions of Tunisia. It is an ecosystem characterized by periodic flooding from subsurface groundwater and fluctuating salinities. Here, we assess the physiological responses and genomic characterization of N. altunense 4.1R to UV-C radiation, as well as osmotic and oxidative stresses. Results showed that the 4.1R strain is able to survive up to 36% of salinity, up to 180 J/m2 to UV-C radiation, and at 50 mM of H2O2, a resistance profile similar to Halobacterium salinarum, a strain often used as UV-C resistant model. In order to understand the genetic determinants of N. altunense 4.1R survival strategy, we sequenced and analyzed its genome. Results showed multiple gene copies of osmotic stress, oxidative stress, and DNA repair response mechanisms supporting its survivability at extreme salinities and radiations. Indeed, the 3D molecular structures of seven proteins related to responses to UV-C radiation (excinucleases UvrA, UvrB, and UvrC, and photolyase), saline stress (trehalose-6-phosphate synthase OtsA and trehalose-phosphatase OtsB), and oxidative stress (superoxide dismutase SOD) were constructed by homology modeling. This study extends the abiotic stress range for the species N. altunense and adds to the repertoire of UV and oxidative stress resistance genes generally known from haloarchaeon.

Testudinimyces gracilis gen. nov, sp. nov. and Astrotestudinimyces divisus gen. nov, sp. nov., two novel, deep-branching anaerobic gut fungal genera from tortoise faeces.

The anaerobic gut fungi (AGF, Neocallimastigomycota) represent a basal zoosporic phylum within the kingdom Fungi. 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 the Neocallimastigomycota. 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 (Testudinimyces and Astrotestudinimyces), and species (T. gracilis and A. divisus) within the order Neocallimastigales. The type species are strains T130AT (T. gracilis) and B1.1T (A. divisus).

Phylogenomic analysis of the Neocallimastigomycota: proposal of Caecomycetaceae fam. nov., Piromycetaceae fam. nov., and emended description of the families Neocallimastigaceae and Anaeromycetaceae.

The anaerobic gut fungi (AGF) represent a coherent phylogenetic clade within the Mycota. Twenty genera have been described so far. Currently, the phylogenetic and evolutionary relationships between AGF genera remain poorly understood. Here, we utilized 52 transcriptomic datasets from 14 genera to resolve AGF inter-genus relationships using phylogenomics, and to provide a quantitative estimate (amino acid identity, AAI) for intermediate rank assignments. We identify four distinct supra-genus clades, encompassing all genera producing polyflagellated zoospores, bulbous rhizoids, the broadly circumscribed genus Piromyces, and the Anaeromyces and affiliated genera. We also identify the genus Khoyollomyces as the earliest evolving AGF genus. Concordance between phylogenomic outputs and RPB1 and D1/D2 LSU, but not RPB2, MCM7, EF1α or ITS1, phylogenies was observed. We combine phylogenomic analysis and AAI outputs with informative phenotypic traits to propose accommodating 14/20 AGF genera into four families: Caecomycetaceae fam. nov. (encompassing the genera Caecomyces and Cyllamyces), Piromycetaceae fam. nov. (encompassing the genus Piromyces), emend the description of the family Neocallimastigaceae to encompass the genera Neocallimastix, Orpinomyces, Pecoramyces, Feramyces, Ghazallomyces, Aestipascuomyces and Paucimyces, as well as the family Anaeromycetaceae to include the genera Oontomyces, Liebetanzomyces and Capellomyces in addition to Anaeromyces. We refrain from proposing families for the deeply branching genus Khoyollomyces and for genera with uncertain position (Buwchfawromyces, Joblinomyces, Tahromyces, Agriosomyces and Aklioshbomyces) pending availability of additional isolates and sequence data; and these genera are designated as 'genera incertae sedis' in the order Neocallimastigales. Our results establish an evolutionary-grounded Linnaean taxonomic framework for the AGF, provide quantitative estimates for rank assignments, and demonstrate the utility of RPB1 as an additional informative marker in Neocallimastigomycota taxonomy.

Taxonomy of the anaerobic gut fungi (Neocallimastigomycota): a review of classification criteria and description of current taxa.

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.

Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota).

Establishing a solid taxonomic framework is crucial for enabling discovery and documentation efforts. This ensures effective communication between scientists as well as reproducibility of results between laboratories, and facilitates the exchange and preservation of biological material. Such framework can only be achieved by establishing clear criteria for taxa characterization and rank assignment. Within the anaerobic fungi (phylum Neocallimastigomycota), the need for such criteria is especially vital. Difficulties associated with their isolation, maintenance and long-term storage often result in limited availability and loss of previously described taxa. To this end, we provide here a list of morphological, microscopic, phylogenetic and phenotypic criteria for assessment and documentation when characterizing newly obtained Neocallimastigomycota isolates. We also recommend a polyphasic rank-assignment scheme for novel genus-, species- and strain-level designations for newly obtained Neocallimastigomycota isolates.

Genomic characterization of three novel Desulfobacterota classes expand the metabolic and phylogenetic diversity of the phylum.

We report on the genomic characterization of three novel classes in the phylum Desulfobacterota. One class (proposed name Candidatus 'Anaeroferrophillalia') was characterized by heterotrophic growth capacity, either fermentatively or utilizing polysulfide, tetrathionate or thiosulfate as electron acceptors. In the absence of organic carbon sources, autotrophic growth via the Wood-Ljungdahl (WL) pathway and using hydrogen or Fe(II) as an electron donor is also inferred for members of the 'Anaeroferrophillalia'. The second class (proposed name Candidatus 'Anaeropigmentia') was characterized by its capacity for growth at low oxygen concentration, and the capacity to synthesize the methyl/alkyl carrier CoM, an ability that is prevalent in the archaeal but rare in the bacterial domain. Pigmentation is inferred from the capacity for carotenoid (lycopene) production. The third class (proposed name Candidatus 'Zymogenia') was characterized by fermentative heterotrophic growth capacity, broad substrate range and the adaptation of some of its members to hypersaline habitats. Analysis of the distribution pattern of all three classes showed their occurrence as rare community members in multiple habitats, with preferences for anaerobic terrestrial, freshwater and marine environments over oxygenated (e.g. pelagic ocean and agricultural land) settings. Special preference for some members of the class Candidatus 'Zymogenia' for hypersaline environments such as hypersaline microbial mats and lagoons was observed.

carP, encoding a Ca2+-regulated putative phytase, is evolutionarily conserved in Pseudomonas aeruginosa and has potential as a biomarker.

Pseudomonas aeruginosa infects patients with cystic fibrosis, burns, wounds and implants. Previously, our group showed that elevated Ca2+ positively regulates the production of several virulence factors in P. aeruginosa, such as biofilm formation, production of pyocyanin and secreted proteases. We have identified a Ca2+-regulated ß-propeller putative phytase, CarP, which is required for Ca2+ tolerance, regulation of the intracellular Ca2+ levels, and plays a role in Ca2+ regulation of P. aeruginosa virulence. Here, we studied the conservation of carP sequence and its occurrence in diverse phylogenetic groups of bacteria. In silico analysis revealed that carP and its two paralogues PA2017 and PA0319 are primarily present in P. aeruginosa and belong to the core genome of the species. We identified 155 single nucleotide alterations within carP, 42 of which lead to missense mutations with only three that affected the predicted 3D structure of the protein. PCR analyses with carP-specific primers detected P. aeruginosa specifically in 70 clinical and environmental samples. Sequence comparison demonstrated that carP is overall highly conserved in P. aeruginosa isolated from diverse environments. Such evolutionary preservation of carP illustrates its importance for P. aeruginosa adaptations to diverse environments and demonstrates its potential as a biomarker.

Genomic Analysis of Family UBA6911 (Group 18 Acidobacteria) Expands the Metabolic Capacities of the Phylum and Highlights Adaptations to Terrestrial Habitats.

Approaches for recovering and analyzing genomes belonging to novel, hitherto-unexplored bacterial lineages have provided invaluable insights into the metabolic capabilities and ecological roles of yet-uncultured taxa. The phylum Acidobacteria is one of the most prevalent and ecologically successful lineages on Earth, yet currently, multiple lineages within this phylum remain unexplored. Here, we utilize genomes recovered from Zodletone Spring, an anaerobic sulfide and sulfur-rich spring in southwestern Oklahoma, as well as from multiple disparate soil and nonsoil habitats, to examine the metabolic capabilities and ecological role of members of family UBA6911 (group 18) Acidobacteria. The analyzed genomes clustered into five distinct genera, with genera Gp18_AA60 and QHZH01 recovered from soils, genus Ga0209509 from anaerobic digestors, and genera Ga0212092 and UBA6911 from freshwater habitats. All genomes analyzed suggested that members of Acidobacteria group 18 are metabolically versatile heterotrophs capable of utilizing a wide range of proteins, amino acids, and sugars as carbon sources, possess respiratory and fermentative capacities, and display few auxotrophies. Soil-dwelling genera were characterized by larger genome sizes, higher numbers of CRISPR loci, an expanded carbohydrate active enzyme (CAZyme) machinery enabling debranching of specific sugars from polymers, possession of a C1 (methanol and methylamine) degradation machinery, and a sole dependence on aerobic respiration. In contrast, nonsoil genomes encoded a more versatile respiratory capacity for oxygen, nitrite, sulfate, and trimethylamine N-oxide (TMAO) respiration, as well as the potential for utilizing the Wood-Ljungdahl (WL) pathway as an electron sink during heterotrophic growth. Our results not only expand our knowledge of the metabolism of a yet-uncultured bacterial lineage but also provide interesting clues on how terrestrialization and niche adaptation drive metabolic specialization within the Acidobacteria. IMPORTANCE Members of the Acidobacteria are important players in global biogeochemical cycles, especially in soils. A wide range of acidobacterial lineages remain currently unexplored. We present a detailed genomic characterization of genomes belonging to family UBA6911 (also known as group 18) within the phylum Acidobacteria. The genomes belong to different genera and were obtained from soil (genera Gp18_AA60 and QHZH01), freshwater habitats (genera Ga0212092 and UBA6911), and an anaerobic digestor (genus Ga0209509). While all members of the family shared common metabolic features, e.g., heterotrophic respiratory abilities, broad substrate utilization capacities, and few auxotrophies, distinct differences between soil and nonsoil genera were observed. Soil genera were characterized by expanded genomes, higher numbers of CRISPR loci, a larger carbohydrate active enzyme (CAZyme) repertoire enabling monomer extractions from polymer side chains, and methylotrophic (methanol and methylamine) degradation capacities. In contrast, nonsoil genera encoded more versatile respiratory capacities for utilizing nitrite, sulfate, TMAO, and the WL pathway, in addition to oxygen as electron acceptors. Our results not only broaden our understanding of the metabolic capacities within the Acidobacteria but also provide interesting clues on how terrestrialization shaped Acidobacteria evolution and niche adaptation.

Genomes of Novel Myxococcota Reveal Severely Curtailed Machineries for Predation and Cellular Differentiation.

Cultured Myxococcota are predominantly aerobic soil inhabitants, characterized by their highly coordinated predation and cellular differentiation capacities. Little is currently known regarding yet-uncultured Myxococcota from anaerobic, nonsoil habitats. We analyzed genomes representing one novel order (o__JAFGXQ01) and one novel family (f__JAFGIB01) in the Myxococcota from an anoxic freshwater spring (Zodletone Spring) in Oklahoma, USA. Compared to their soil counterparts, anaerobic Myxococcota possess smaller genomes and a smaller number of genes encoding biosynthetic gene clusters (BGCs), peptidases, one- and two-component signal transduction systems, and transcriptional regulators. Detailed analysis of 13 distinct pathways/processes crucial to predation and cellular differentiation revealed severely curtailed machineries, with the notable absence of homologs for key transcription factors (e.g., FruA and MrpC), outer membrane exchange receptor (TraA), and the majority of sporulation-specific and A-motility-specific genes. Further, machine learning approaches based on a set of 634 genes informative of social lifestyle predicted a nonsocial behavior for Zodletone Myxococcota. Metabolically, Zodletone Myxococcota genomes lacked aerobic respiratory capacities but carried genes suggestive of fermentation, dissimilatory nitrite reduction, and dissimilatory sulfate-reduction (in f_JAFGIB01) for energy acquisition. We propose that predation and cellular differentiation represent a niche adaptation strategy that evolved circa 500 million years ago (Mya) in response to the rise of soil as a distinct habitat on Earth. IMPORTANCE The phylum Myxococcota is a phylogenetically coherent bacterial lineage that exhibits unique social traits. Cultured Myxococcota are predominantly aerobic soil-dwelling microorganisms that are capable of predation and fruiting body formation. However, multiple yet-uncultured lineages within the Myxococcota have been encountered in a wide range of nonsoil, predominantly anaerobic habitats, and the metabolic capabilities, physiological preferences, and capacity of social behavior of such lineages remain unclear. Here, we analyzed genomes recovered from a metagenomic analysis of an anoxic freshwater spring in Oklahoma, USA, that represent novel, yet-uncultured, orders and families in the Myxococcota. The genomes appear to lack the characteristic hallmarks for social behavior encountered in Myxococcota genomes and displayed a significantly smaller genome size and a smaller number of genes encoding biosynthetic gene clusters, peptidases, signal transduction systems, and transcriptional regulators. Such perceived lack of social capacity was confirmed through detailed comparative genomic analysis of 13 pathways associated with Myxococcota social behavior, as well as the implementation of machine learning approaches to predict social behavior based on genome composition. Metabolically, these novel Myxococcota are predicted to be strict anaerobes, utilizing fermentation, nitrate reduction, and dissimilarity sulfate reduction for energy acquisition. Our results highlight the broad patterns of metabolic diversity within the yet-uncultured Myxococcota and suggest that the evolution of predation and fruiting body formation in the Myxococcota has occurred in response to soil formation as a distinct habitat on Earth.

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.

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.

Genomic Characterization of Candidate Division LCP-89 Reveals an Atypical Cell Wall Structure, Microcompartment Production, and Dual Respiratory and Fermentative Capacities.

Recent experimental and bioinformatic advances enable the recovery of genomes belonging to yet-uncultured microbial lineages directly from environmental samples. Here, we report on the recovery and characterization of single amplified genomes (SAGs) and metagenome-assembled genomes (MAGs) representing candidate phylum LCP-89, previously defined based on 16S rRNA gene sequences. Analysis of LCP-89 genomes recovered from Zodletone Spring, an anoxic spring in Oklahoma, predicts slow-growing, rod-shaped organisms. LCP-89 genomes contain genes for cell wall lipopolysaccharide (LPS) production but lack the entire machinery for peptidoglycan biosynthesis, suggesting an atypical cell wall structure. The genomes, however, encode S-layer homology domain-containing proteins, as well as machinery for the biosynthesis of CMP-legionaminate, inferring the possession of an S-layer glycoprotein. A nearly complete chemotaxis machinery coupled to the absence of flagellar synthesis and assembly genes argues for the utilization of alternative types of motility. A strict anaerobic lifestyle is predicted, with dual respiratory (nitrite ammonification) and fermentative capacities. Predicted substrates include a wide range of sugars and sugar alcohols and a few amino acids. The capability of rhamnose metabolism is confirmed by the identification of bacterial microcompartment genes to sequester the toxic intermediates generated. Comparative genomic analysis identified differences in oxygen sensitivities, respiratory capabilities, substrate utilization preferences, and fermentation end products between LCP-89 genomes and those belonging to its four sister phyla (Calditrichota, SM32-31, AABM5-125-24, and KSB1) within the broader FCB (Fibrobacteres-Chlorobi-Bacteroidetes) superphylum. Our results provide a detailed characterization of members of the candidate division LCP-89 and highlight the importance of reconciling 16S rRNA-based and genome-based phylogenies.IMPORTANCE Our understanding of the metabolic capacities, physiological preferences, and ecological roles of yet-uncultured microbial phyla is expanding rapidly. Two distinct approaches are currently being utilized for characterizing microbial communities in nature: amplicon-based 16S rRNA gene surveys for community characterization and metagenomics/single-cell genomics for detailed metabolic reconstruction. The occurrence of multiple yet-uncultured bacterial phyla has been documented using 16S rRNA surveys, and obtaining genome representatives of these yet-uncultured lineages is critical to our understanding of the role of yet-uncultured organisms in nature. This study provides a genomics-based analysis highlighting the structural features and metabolic capacities of a yet-uncultured bacterial phylum (LCP-89) previously identified in 16S rRNA surveys for which no prior genomes have been described. Our analysis identifies several interesting structural features for members of this phylum, e.g., lack of peptidoglycan biosynthetic machinery and the ability to form bacterial microcompartments. Predicted metabolic capabilities include degradation of a wide range of sugars, anaerobic respiratory capacity, and fermentative capacities. In addition to the detailed structural and metabolic analysis provided for candidate division LCP-89, this effort represents an additional step toward a unified scheme for microbial taxonomy by reconciling 16S rRNA gene-based and genomics-based taxonomic outlines.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

Global Distribution Patterns and Pangenomic Diversity of the Candidate Phylum "Latescibacteria" (WS3).

We investigated the global distribution patterns and pangenomic diversity of the candidate phylum "Latescibacteria" (WS3) in 16S rRNA gene as well as metagenomic data sets. We document distinct distribution patterns for various "Latescibacteria" orders in 16S rRNA gene data sets, with prevalence of orders sediment_1 in terrestrial, PBSIII_9 in groundwater and temperate freshwater, and GN03 in pelagic marine, saline-hypersaline, and wastewater habitats. Using a fragment recruitment approach, we identified 68.9 Mb of "Latescibacteria"-affiliated contigs in publicly available metagenomic data sets comprising 73,079 proteins. Metabolic reconstruction suggests a prevalent saprophytic lifestyle in all "Latescibacteria" orders, with marked capacities for the degradation of proteins, lipids, and polysaccharides predominant in plant, bacterial, fungal/crustacean, and eukaryotic algal cell walls. As well, extensive transport and central metabolic pathways for the metabolism of imported monomers were identified. Interestingly, genes and domains suggestive of the production of a cellulosome-e.g., protein-coding genes harboring dockerin I domains attached to a glycosyl hydrolase and scaffoldin-encoding genes harboring cohesin I and CBM37 domains-were identified in order PBSIII_9, GN03, and MSB-4E2 fragments recovered from four anoxic aquatic habitats; hence extending the cellulosomal production capabilities in Bacteria beyond the Gram-positive Firmicutes In addition to fermentative pathways, a complete electron transport chain with terminal cytochrome c oxidases Caa3 (for operation under high oxygen tension) and Cbb3 (for operation under low oxygen tension) were identified in PBSIII_9 and GN03 fragments recovered from oxygenated and partially/seasonally oxygenated aquatic habitats. Our metagenomic recruitment effort hence represents a comprehensive pangenomic view of this yet-uncultured phylum and provides insights broader than and complementary to those gained from genome recovery initiatives focusing on a single or few sampled environments.IMPORTANCE Our understanding of the phylogenetic diversity, metabolic capabilities, and ecological roles of yet-uncultured microorganisms is rapidly expanding. However, recent efforts mainly have been focused on recovering genomes of novel microbial lineages from a specific sampling site, rather than from a wide range of environmental habitats. To comprehensively evaluate the genomic landscape, putative metabolic capabilities, and ecological roles of yet-uncultured candidate phyla, efforts that focus on the recovery of genomic fragments from a wide range of habitats and that adequately sample the intraphylum diversity within a specific target lineage are needed. Here, we investigated the global distribution patterns and pangenomic diversity of the candidate phylum "Latescibacteria" Our results document the preference of specific "Latescibacteria" orders to specific habitats, the prevalence of plant polysaccharide degradation abilities within all "Latescibacteria" orders, the occurrence of all genes/domains necessary for the production of cellulosomes within three "Latescibacteria" orders (GN03, PBSIII_9, and MSB-4E2) in data sets recovered from anaerobic locations, and the identification of the components of an aerobic respiratory chain, as well as occurrence of multiple O2-dependent metabolic reactions in "Latescibacteria" orders GN03 and PBSIII_9 recovered from oxygenated habitats. The results demonstrate the value of phylocentric pangenomic surveys for understanding the global ecological distribution and panmetabolic abilities of yet-uncultured microbial lineages since they provide broader and more complementary insights than those gained from single-cell genomic and/or metagenomic-enabled genome recovery efforts focusing on a single sampling site.

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).

Microbial community structure analysis of a benzoate-degrading halophilic archaeal enrichment.

A benzoate-degrading archaeal enrichment was developed using sediment samples from Rozel Point at Great Salt Lake, UT. The enrichment degraded benzoate as the sole carbon source at salinity ranging from 2.0 to 5.0 M NaCl with highest rate of degradation observed at 4.0 M. The enrichment was also tested for its ability to grow on other aromatic compounds such as 4-hydroxybenzoic acid (4-HBA), gentisic acid, protocatechuic acid (PCA), catechol, benzene and toluene as the sole sources of carbon and energy. Of these, the culture only utilized 4-HBA as the carbon source. To determine the initial steps in benzoate degradation pathway, a survey of ring-oxidizing and ring-cleaving genes was performed using degenerate PCR primers. Results showed the presence of 4-hydroxybenzoate 3-monooxygenase (4-HBMO) and protocatechuate 3, 4-dioxygenase (3,4-PCA) genes suggesting that the archaeal enrichment might degrade benzoate to 4-HBA that is further converted to PCA by 4-HBMO and, thus, formed PCA would undergo ring-cleavage by 3,4-PCA to form intermediates that enter the Krebs cycle. Small subunit rRNA gene-based diversity survey revealed that the enrichment consisted entirely of class Halobacteria members belonging to the genera Halopenitus, Halosarcina, Natronomonas, Halosimplex, Halorubrum, Salinarchaeum and Haloterrigena. Of these, Halopenitus was the dominant group accounting for almost 91 % of the total sequences suggesting their potential role in degrading oxygenated aromatic compounds at extreme salinity.