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.

Murphy CL ，Youssef NH ，Hanafy RA ，Couger MB ，Stajich JE ，Wang Y ，Baker K ，Dagar SS ，Griffith GW ，Farag IF ，Callaghan TM ，Elshahed MS ... -  《-》

Molecular Dating of the Emergence of Anaerobic Rumen Fungi and the Impact of Laterally Acquired Genes.

The anaerobic gut fungi (AGF), or Neocallimastigomycota, inhabit the rumen and alimentary tract of herbivorous mammals, where they play important roles in the degradation of plant fiber. Comparative genomic and phylogenomic analyses of the AGF have long been hampered by their fastidious growth condition, as well as their large (up to 200 Mb) and AT-biased (78 to 84%) genomes. We sequenced 21 AGF transcriptomes and combined them with 5 available AGF genome sequences to explore their evolutionary relationships, time their divergence, and characterize gene gain/loss patterns associated with their evolution. We estimate that the most recent common ancestor of the AGF diverged 66 (±10) million years ago, a time frame that coincides with the evolution of grasses (Poaceae), as well as the mammalian transition from insectivory to herbivory. The concordance of independent estimations suggests that AGF have been important in shaping the success of mammalian herbivory transition by improving the efficiency of energy acquisition from recalcitrant plant materials. Comparative genomics identified multiple lineage-specific genes in the AGF, two of which were acquired from rumen gut bacteria and animal hosts via horizontal gene transfer (HGT). A third AGF domain, plant-like polysaccharide lyase, represents a novel gene in fungi that potentially aids AGF to degrade pectin. Analysis of genomic and transcriptomic sequences confirmed both the presence and expression of these lineage-specific genes in nearly all AGF clades. These genetic elements may contribute to the exceptional abilities of AGF to degrade plant biomass and enable metabolism of the rumen microbes and animal hosts.IMPORTANCE Anaerobic fungi living in the rumen of herbivorous mammals possess an extraordinary ability to degrade plant biomass. We examined the origin and genomic composition of these poorly characterized anaerobic gut fungi using both transcriptome and genomic data. Phylogenomics and molecular dating analyses found remarkable concurrence of the divergence times of the rumen fungi, the forage grasses, and the dietary shift of ancestral mammals from primarily insectivory to herbivory. Comparative genomics identified unique machinery in these fungi to utilize plant polysaccharides. The rumen fungi were also identified with the ability to code for three protein domains with putative functions in plant pectin degradation and microbial defense, which were absent from all other fungal organisms (examined over 1,000 fungal genomes). Two of these domains were likely acquired from rumen gut bacteria and animal hosts separately via horizontal gene transfer. The third one is a plant-like polysaccharide lyase, representing a unique fungal enzyme with potential pectin breakdown abilities.

Wang Y ，Youssef NH ，Couger MB ，Hanafy RA ，Elshahed MS ，Stajich JE ... -  《-》

Seven new Neocallimastigomycota genera from wild, zoo-housed, and domesticated herbivores greatly expand the taxonomic diversity of the phylum.

Hanafy RA ，Lanjekar VB ，Dhakephalkar PK ，Callaghan TM ，Dagar SS ，Griffith GW ，Elshahed MS ，Youssef NH ... -  《-》

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.

Pratt CJ ，Meili CH ，Jones AL ，Jackson DK ，England EE ，Wang Y ，Hartson S ，Rogers J ，Elshahed MS ，Youssef NH ... -  《Nature Communications》

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

Hanafy RA ，Elshahed MS ，Liggenstoffer AS ，Griffith GW ，Youssef NH ... -  《MYCOLOGIA》