DNA sequencing of the 31 gastrointestinal digesta samples resulted in 4,923,858 (mean of 158,834 ± 76,389) forward reads (R1), which were denoised using deblur into 2,524 amplicon sequence variants (ASVs).
Microbial biogeography through the digestive tract of two wombat species
The stomach and small intestine of hindgut fermenting mammals are harsh environments for microbes to survive, typified by extremely low pH and various digestive enzymes and bile salts respectively. The colon by contrast is a more habitable region for complex gut microbial communities to thrive, and where the bulk of microbial fermentation of plant material is thought to occur. To test if these different gut regions influenced the microbial communities, we measured microbial richness (the number of ASVs) of duplicate luminal samples taken throughout the GI tract of each wombat species. Microbial richness tended to increase through the length of the digestive tract for both wombat species (Fig. 1). Stomach and small intestine samples had the lowest ASV richness (~ 100–200), followed by the first two proximal colon samples (PC1/PC2: ~400–500), with the latter proximal colon and distal colon samples exhibiting the highest ASV richness (~ 500–800).
Like richness, the types of microorganisms living in a site and how they are structured (composition) can also be influenced by the environmental factors of that site. To test this, we calculated the microbial compositions of these distinct sites using both the abundance weighted and unweighted UniFrac distance metrics. Using both methods, the major differences observed in microbial composition were associated with GI tract site not wombat species, with stomach, small intestine colon, and colon samples clustering across the axis of most variation (Axis 1: Fig. 2A/B). Colon samples from the different wombat species were separated across axis 2 for the unweighted UniFrac analysis (Fig. 2A), whereas axis 2 of the abundance-weighted analysis further separated proximal and distal colon samples (Fig. 2B). Taking into account colon samples only, species-specific differences in abundance-weighted microbial composition between colon samples were observed across axis 2 (SI Fig. 1B). Interestingly, the microbial composition of the first proximal colon samples of different wombat species are more similar to each other than they are to the distal colon samples of the same species (Fig. 2B, SI Fig. 2B).
We next investigated what types of microbes (the taxonomic composition) were present in the different GI samples for both wombat species. Consistent with differences we observed in the ordination analyses, the stomach and small intestine samples were taxonomically distinct to the colon samples for both wombat species (Fig. 3). At the phylum level, these samples tended to be dominated by Proteobacteria, Firmicutes, and Fusobacteriota (SI Fig. 3), with the dominant families being Pasteurellaceae (59% in BNW-ST, 56% in SHNW-ST, 43% in SHNW-PSI), Peptostreptococcaceae (67% in BNW-SI), and Fusobacteriaceae (81% in SHNW-DSI) (Fig. 3). In contrast, the colon samples for both wombat species were dominated by the Bacteroidota, Firmicutes, and Spirochaetota phyla (SI Fig. 3). We also observed taxonomic differences between proximal and distal colon samples for both the wombat species (Fig. 3). The first proximal colon samples were dominated by Prevotellaceae (~ 40% relative abundance), and Fibrobacteraceae (~ 10% for the BNW), whereas distal colon samples contained higher proportions of Spirochaetaceae, Rikenellaceae, and WCHB1-41. These results indicate that microbial diversity and composition vary throughout the GI tract of two hindgut fermenting species, and that the start of the proximal colon–the putative primary site of fermentation in these wombat species–is distinct from the distal colon.
Microbial differences between proximal and distal colon sites
We next focused on characterising microbial community compositional differences between the proximal and distal colons for both species. As there were many ASVs that were classified to the same taxa we collapsed the ASV table to the genus-level, and ran ANCOM-II to identify genera that were differentially abundant between different regions of the colon (i.e. PC1, PC2, DC, etc.). Of the 255 genera classified in the dataset, ANCOM-II identified 84 that were significantly differentially abundant throughout the colon when the W-statistic threshold for rejecting the null was at 70% (Fig. 4) (SI Table 1). Taxa that had significantly higher abundance in PC1/2 vs. DC include: Prevotella (W = 245), Prevotellaceae_UCG-001 (W = 241), Bacteroides (W = 239), and Bacteroidales_RF16_group (W = 231). Taxa with a higher relative abundance in DC vs. PC1/2 include: Bacteroidales_BS11_gut_group (W = 250), Bacteroidales_UCG-001 (W = 243), WCHB1-41 (W = 242), and Izemoplasmatales (W = 226).
To test how representative the microbial community of faeces are to the first proximal colon site, we measured how many ASVs were shared between PC1 and the last DC sample for each wombat species. Surprisingly, only 99 (10.6%) and 204 (18.7%) ASVs were shared between PC1 and DC samples for the BNW and SHNW, respectively (Fig. 5). See (SI Fig. 3) for a cross-species comparison. The ASVs that were unique to the BNW PC1 and DC sites accounted for 25% and 64% of the relative abundance in those sites, respectively. Likewise, the ASVs that were unique to the SHNW PC1 and DC sites accounted for 32% and 67%, respectively. These results indicate that the first proximal colon and distal colon sites harbour distinct communities of microbes.