File S1 - Metagenomic Insights into Metabolic Capacities of the Gut Microbiota in a Fungus-Cultivating Termite (Odontotermes yunnanensis)

Figure S1. DGGE fingerprint of the V3 region of 16S rRNA genes amplified from termite gut eDNA prepared with two different fractionation procedures. V3 region of 16S rRNA genes was amplified with primers Eubac7 Vf-GC/Vr from 10 ng of eDNA prepared by 1: directly homogenizing whole gut tissues with a 2-ml Tenbroeck tissue grinder, and centrifugate at 200×g to remove coarse particles; 2: homogenizing with pipette after trypsin digestion, and centrifugating at 800×g to extrude eukaryotic cells. Detailed procedures for DGGE could be seen in our previous work of preparing gut eDNA for M. annandalei [14]. Figure S2. Distribution of 454 sequences of the whole gut metagenome of O. yunnanensis. Figure S3. Collectors’ curves (Collector, Chao1, and ACE) derived from full-length 16S RNA gene library of O. yunnanensis whole gut metagenome. Phylotype cutoffs were 97%, 98%, and 99%, respectively. Figure S4. Statistically different SEED classifications between the gut microbiomes of O. yunnanensis and the Nasutitermes sp. [15]. Classifications statistically overrepresented in the Odontotermes metagenome were marked with blue circles, while those statistically overrepresented in the Nasutitermes metagenome were marked with orange ones (P<0.05 and Ratio of proportions >1.1 were shown). SEED subsystems-based annotation of both metagenomes was performed as described in the methods. The proportions of environmental gene tags (EGTs) in each classification with respect to the total number of SEED annotated ones in individual metagenome were calculated, based on which ratio of proportions of each classification in the two datasets were further calculated. Gene-centric statistic analysis was performed with two-sided Fisher’s exact test implemented in the STAMP program [33]. P values were corrected by the Benjamini-Hochberg multiple test and confidence intervals were calculated by the Asymptotic method. Figure S5. Subsystem distributions in partial statistically different SEED classifications between the gut microbiomes of O. yunnanensis and the Nasutitermes sp. [15] Subsystem distribution of motility and chemotaxis (A), phages, prophages, transposable elements, plasmids (B), metabolism of aromatic compounds (C), and nitrogen metabolism (D). Noticeably, in (C) the two subsystems statistically overrepresented in the Nasutitermes metagenome both belong to peripheral pathways of catabolism of aromatic compounds (labeled in red caption), while all subsystems statistically enriched in the Odontotermes metagenome belong to metabolism of central aromatic intermediates or aromatic compounds (labeled in black caption, also see Table S5 in File S1). Statistic analysis was performed with the same procedures and parameters as for Figure S4. Figure S6. Electrophoresis detection of the PCR amplification product of the nifH Gene. The highly degenerate primers (Pf: 5′-TGXGAXCCYAAZGCYGA-3′ X = T or C, Y = A,C,G, or T and Pr: 5′-AWYGCCATCATXTCYCC-3′ Z = A or G, W = A,T, or G) designed by Kirshtein et al [57]. were used to amplify the ∼360 bp fragment of the nifH gene, which encodes the iron protein of nitrogenase that catalyzes N₂ fixation. M, Quick-Load 100 bp DNA Ladder; 1, amplification products from the whole gut metagenomic DNA of the higher wood-feeding Globitermes brachycerastes; 2, amplification products from the whole gut metagenomic DNA of the fungus-cultivating O. yunnanensis; 3, amplification products from the whole gut metagenomic DNA of the fungus-cultivating Macrotermes annandelei. It revealed that the ∼360 bp fragment of the nifH gene which encodes the iron protein of nitrogenase could only be amplified from the wood-feeding higher termite species. Table S1. Phylotype representatives of 16S rRNA sequences obtained from clone library in the whole gut microbiome of Odontotermes yunnanensis. Table S2. Carbohydrate-active gene modules detected in the gut metagenome of O.yunnanensis. Table S3. Domains often associated with GH catalytic domains detected in the gut metagenome of O.yunnanensis. Table S4. Comparison of CAZy profiles of the whole gut metagenome of O.yunnanensis with those of the leaf cutter ant fungus garden [18], wallaby foregut [17], and the wood-feeding Nasutitermes sp. hindgut [15]. Table S5. Distribution of statistically different subsystems in metabolism of aromatic compounds between the gut microbiomes of O. yunnanensis and the Nasutitermes sp. Table S6. Composition of statistically different subsystems in nitrogen metabolism between the gut microbiomes of O. yunnanensis and the Nasutitermes sp.

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