Precise Fecal Microbiome of the Herbivorous Tibetan Antelope Inhabiting High-Altitude Alpine Plateau

Abstract

The metataxonomic approach combining 16S rRNA gene amplicon sequencing using the PacBio Technology with the application of the operational phylogenetic unit (OPU) approach, has been used to analyze the fecal microbial composition of the high-altitude and herbivorous Tibetan antelopes. The fecal samples of the antelope were collected in Hoh Xil National Nature Reserve, at an altitude over 4500 m, the largest depopulated zone in Qinghai-Tibetan Plateau, China, where non-native animals or humans may experience life-threatening acute mountain sickness. In total, 104 antelope fecal samples were enrolled in this study, and were clustered into 61,258 operational taxonomic units (OTUs) at an identity of 98.7% and affiliated with 757 OPUs, including 144 known species, 256 potentially new species, 103 potentially higher taxa within known lineages. In addition, 254 comprised sequences not affiliating with any known family, and the closest relatives were unclassified lineages of existing orders or classes. A total of 42 out of 757 OPUs conformed to the core fecal microbiome, of which four major lineages, namely, un-cultured Ruminococcaceae, Lachnospiraceae, Akkermansia, and Christensenellaceae were associated with human health or longevity. The current study reveals that the fecal core microbiome of antelope is mainly composited of uncultured bacteria. The most abundant core taxa, namely, uncultured Ruminococcaceae, uncultured Akkermansia, uncultured Bacteroides, uncultured Christensenellaceae, uncultured Mollicutes, and uncultured Lachnospiraceae, may represent new bacterial candidates at high taxa levels, and several may have beneficial roles in health promotion or anti-intestinal dysbiosis. These organisms should be further isolated and evaluated for potential effect on human health and longevity.

Keywords: Tibetan antelope; full-length 16S rRNA gene; herbivorous; high-altitude; metataxonomics; microbiome; operational phylogenetic unit.

FIGURE 1

Proportion of uncultured bacteria in the feces of Tibetan antelope. (A) The proportion of reads. The number of reads classified into uncultured bacteria, bacteria at higher taxa levels, new species, and known species was 827,656, 31,743, 6,970, and 1,065, respectively. (B) The proportion of OPUs. The number of OPUs that classified into uncultured bacteria, higher taxa levels, new species and known species was 254, 103, 256, and 144, respectively.

FIGURE 2

Profiles of known species. (A) Taxonomic tree of 144 known species. Each dot represents a taxonomic entity. From the inner to the outer circles, the taxonomic levels range from phylum to species. Dots of different colors indicate different taxonomic levels according to the color key shown. Numbers in parentheses indicate the total number of unique taxonomies detected at each level. The most abundant species, which were composed of more than 10 reads are denoted by asterisk. Species in pink indicate those having medical significance, among which the top 20 species, which were composed of more than 10 reads are denoted by pink asterisk. (B) Sequences numbers of the top 20 known species having medical significance.

FIGURE 3

Profiles of new species. (A) Taxonomic tree of 256 new species. The most abundant species, which were composed of more than 50 reads are showed by pink asterisk. (B) The top 18 abundant new species with more than 50 reads.

FIGURE 4

Taxonomic tree of uncultured bacteria. Each dot represents a taxonomic entity. From the inner to the outer circles, the taxonomic levels range from phylum to genus. Dots of different colors indicate different taxonomic levels according to the color key shown. The outermost taxa of each OPU show the highest taxa level it can reach. Numbers in parentheses indicate the total number of unique taxonomies detected at each level. The core OPUs shared by all antelopes are showed by asterisk.

FIGURE 5

Abundances of core bacterial taxa shared by all samples. Sequences numbers of these predominant taxa from left to right were 329,996, 85,042, 73,443, 72,199, 44,772, 43,430, 21,233, 20,151, 17,182, 9,182, 7,041, 6,865, 6,084, 4,630, 4,135, 4,050, 2,302, and 1,576, respectively.

FIGURE 6

Phylogeny of core uncultured Mollicutes. A phylogenetic comparison was carried out using the representatives 16S rRNA sequences of OPU 166, which was defined as uncultured Mollicutes, type strains of five orders identified in the Mollicutes class, and two type strains in Firmicutes phylum, which were used as outgroup. OPU 166 is grouped in red box with the total number of reads shown in parentheses. The maximum likelihood phylogeny was constructed using RAxML with 100 replications (Escherichia coli-wide tree) as described in the Section “Materials and Methods.”

FIGURE 7

Phylogeny of core uncultured Bacteroidale. A phylogenetic comparison was carried out using the representative sequences of OPU 33 and type strains of six families identified in the Bacteroidale order. OPU 33 was grouped in the red box with the total number of reads shown in parentheses. The 16S rRNA sequences of Bacteroidaceae from human were downloaded and included in the analysis (http://metagenomics.anl.gov/linkin.cgi?project=17761) to assess reliability of the method we used, which are showed in yellow. The Maximum likelihood phylogeny was constructed using RAxML with 100 replications.