Microbial biogeography of the wombat gastrointestinal tract

Abstract

Most herbivorous mammals have symbiotic microbes living in their gastrointestinal tracts that help with harvesting energy from recalcitrant plant fibre. The bulk of research into these microorganisms has focused on samples collected from faeces, representing the distal region of the gastrointestinal (GI) tract. However, the GI tract in herbivorous mammals is typically long and complex, containing different regions with distinct physico-chemical properties that can structure resident microbial communities. Little work has been done to document GI microbial communities of herbivorous animals at these sites. In this study, we use 16S rRNA gene sequencing to characterize the microbial biogeography along the GI tract in two species of wombats. Specifically, we survey the microbes along four major gut regions (stomach, small intestine, proximal colon, distal colon) in a single bare-nosed wombat (Vombatus ursinus) and a single southern hairy-nosed wombat (Lasiorhinus latifrons). Our preliminary results show that GI microbial communities of wombats are structured by GI region. For both wombat individuals, we observed a trend of increasing microbial diversity from stomach to distal colon. The microbial composition in the first proximal colon region was more similar between wombat species than the corresponding distal colon region in the same species. We found several microbial genera that were differentially abundant between the first proximal colon (putative site for primary plant fermentation) and distal colon regions (which resemble faecal samples). Surprisingly, only 10.6% (98) and 18.8% (206) of amplicon sequence variants (ASVs) were shared between the first proximal colon region and the distal colon region for the bare-nosed and southern hairy-nosed wombat, respectively. These results suggest that microbial communities in the first proximal colon region-the putative site of primary plant fermentation in wombats-are distinct from the distal colon, and that faecal samples may have limitations in capturing the diversity of these communities. While faeces are still a valuable and effective means of characterising the distal colon microbiota, future work seeking to better understand how GI microbiota impact the energy economy of wombats (and potentially other hindgut-fermenting mammals) may need to take gut biogeography into account.

Keywords: Australia; Biogeography; Gastrointestinal; Gut; Marsupials; Microbial Ecology; Microbiome; Wombat; Zoology.

Figure 1. Illustration of wombat gut tracts and samples collected.

Drawn illustrations of Vombatus ursinus (top) and Lasiorhinus latifrons (bottom) gastrointestinal tracts based on Barboza & Hume (1992a). Stars represent locations where duplicate digesta samples were collected for the study. BNW, Bare-nosed wombat; SHNW, Southern hairy-nosed wombat.

Figure 2. Taxonomic composition of the different sample types at the family level.

Only the top 20 most abundant families are displayed for clarity (these families account for 81.17% of reads). The ‘Other’ bin (grey) contains the other families. Replicate samples were merged per sample site. (A) bare-nosed wombat. (B) southern hairy-nosed wombat. The widths of the bars are scaled to the length of the GI region.

Figure 3. Microbial diversity (ASV richness) of samples collected throughout the wombat digestive tract, ordered from start to end.

ST, stomach; SI, small intestine; PSI, proximal small intestine; DSI, distal small intestine; PC, proximal colon; DC, distal colon. Two technical replicates were collected and processed for each site (joined by lines). (A) Bare-nosed wombat. (B) Southern hairy-nosed wombat. Digestive tract drawings were adapted from Barboza & Hume (1992a).

Figure 4. Microbial community structure among sites along the wombat GI tract.

(A) PCoA of unweighted UniFrac distances and (B) PCoA of weighted UniFrac distances. Samples are coloured by sample type, and shaped by host species. Numbers indicate order in which samples occur in the digestive tract. Lines connect samples from the same sample type.

Figure 5. Heat map of the microbial genera that were found to be differentially abundant between proximal and distal colon sites.

The abundance of genera that were found to be differentially abundant (0.7 threshold) in the ANCOM-II analysis are displayed. Black indicates 0 assigned reads. Non-genus assignments are prefixed with the lowest level of taxonomy that could be assigned (e.g., f_– = family). Taxonomy string are prefixed with ‘Phylum –’.

Figure 6. Euler diagram of amplicon sequence variants (ASVs) shared between proximal and distal colon sites for both wombat species.

Euler diagram of amplicon sequence variants (ASVs) shared between proximal colon 1 (PC1) and last distal colon site (DC) for (A) bare-nosed wombat (BNW) and (B) southern hairy-nosed wombat (SHNW). Percentages represent the proportion of ASVs specific to a given area.