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

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

Yan Wang et al. mSystems.

Abstract

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.

Keywords: HGT; comparative genomics; divergence time estimation; evolution; phylogenomics.

Figures

FIG 1
FIG 1
Bayesian phylogenomic maximum clade credibility tree of Neocallimastigomycota with divergence time estimation. All clades are fully supported by Bayesian posterior probabilities (BPP). For clarity, mean ages and 95% highest-probability density ranges (blue bars) are denoted on the nodes above the rank of genus.
FIG 2
FIG 2
Cladogram and heatmap enrichment of the Pfam domains between Neocallimastigomycota and Chytridiomycota. (a) Cladogram showing the phylogenetic relationship of the compared taxa (Neocallimastigomycota genomes are in bold). (b) Heatmap plot of natural logarithm of the domain copy numbers showing the ones uniquely gained in Neocallimastigomycota (Pfam domains highlighted in this study are in blue). (c) Pfam domains highly enriched in Neocallimastigomycota. (d) Pfam domains absent in Neocallimastigomycota (presented domains are partial; see Table S1 for the full list).
FIG 3
FIG 3
Midpoint-rooted phylogenetic tree of the Cthe_2159 domain. All 126 Neocallimastigomycota (AGF) copies (copies that have >90% identities have been removed) form a single clade (red) indicating the HGT donor, Clostridiales bacterium C5EMF8 (an obligate rumen bacterium), with strong support of maximum likelihood bootstrap (98/100). Included bacterial lineages were assigned different colors according to their phylogenetic classification (see legend for detailed information; the complete tree with all tip information is shown in Fig. S3).
FIG 4
FIG 4
Phylogenetic tree of the 83 Cthe_2159 domains identified in five AGF genomes. Coding sequence identifiers used in the original annotation files are labeled with species names in tree tips. The tree is rooted with the closest related bacterial homolog found in Clostridiales bacterium. The domain map on the right shows the conserved domains produced by the Cthe_2159-containing genes.
FIG 5
FIG 5
Phylogenetic tree of the animal-like Gal-Lectin domain identified in Neocallimastigomycota. (a) Collapsed phylogenetic tree based on protein sequences (rooted with the bacterial outgroup), including clades of Neocallimastigomycota (red), animals (blue; three clades are labeled 1 to 3), plants (green), and bacteria (brown) (a complete tree with all tip information is shown in Fig. S4). (b) Schematic diagrams showing the Gal_Lectin and other conserved domains on the same protein in each clade individually (dotted box highlights the aligned region used to produce the phylogenetic tree).
FIG 6
FIG 6
Radial phylogenetic tree of the Rhamnogal_lyase domain encoded by the Neocallimastigomycota (red). Plant copies are colored in green, and other homologous fungal genes are colored in brown. Oomycetes are in cyan, and animal copies only known in the mountain pine beetle Dendroctonus ponderosae are in blue. Bacterial branches are in black. The tree also included homologs of RhgB_N and Rhamnogalacturonan lyase A, B, and C. Domain names are suggested using NCBI’s conserved domain search tool (cutoff 1E−5) with unaligned FASTA sequences (refer to Fig. S6 for a tree with detailed information).

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