3.4 Variation in gut bacterial communities of giant pandas with different lifestyles
A total of 4,747,957 bacterial 16S rRNA gene sequencing genes were obtained from samples in the lifestyle change experiments. After removal of mitochondria and chloroplasts sequences, 4,644,322 16S rRNA gene sequences were clustered into 3,720 OTUs at the 97% sequence identity threshold. The richness and diversity of gut bacterial communities varied with lifestyle shifts and significant differences were observed among the wild-training I, wild-training II, reintroduced and wild groups (Figure 3a, b). The richness and diversity of gut microbial communities from wild pandas were higher than those of wild-training I or wild-training II groups. Specifically, gut bacterial community richness in the wild pandas was significantly higher than those of wild-training I pandas (p < 0.05, ANOVA test). Moreover, community diversity in the wild pandas was significantly higher than those of the wild-training II and reintroduced pandas (p < 0.05, ANOVA test). PCoA of bacterial community composition indicated that samples from hosts with the same lifestyle clustered together and distinctly from others (Figure 3c).
Proteobacteria and Firmicutes were the dominant phyla among lifestyle shift samples regardless of lifestyle, comprising more than 98.0% of the sequences (Figure 3d). In particular, Proteobacteria was the dominant phylum in wild-training I panda gut communities (61.0%) and was significantly higher than in those of other groups (6.4% in wild-training II and 15.2% in reintroduced pandas) (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4) (Figure 3d, e). Conversely, Firmicutes was the dominant phylum in the wild-training II (92.5%) and reintroduced (84.2%) pandas, and was significantly higher than in the communities of wild-training I pandas (37.8%) (Non-parametric factorial Kruskal-Wallis sum-rank tests, LDA>4).
The distributions of the 10 most abundant bacterial genera in each group were further investigated, excluding unidentified genera (Figure 3e, f).Escherichia (30.6%), Acinetobacter (22.4%), andStreptococcus (20.1%) were the most abundant genera in the communities of wild-training I pandas (Figure 3d). Among these,Escherichia abundances were significantly enriched in the communities of the wild-training I group, and significantly lower in the reintroduced group (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4) (Figure 3e). In addition, the abundances ofAcinetobacter in the communities of wild-training I pandas (22.4%) were significantly higher than in those of wild-training II (0.4%) and reintroduced (3.2%) pandas (Non-parametric factorial Kruskal-Wallis sum-rank tests, LDA>4). Streptococcus(64.2%) was the dominant genus in wild-training II panda communities and were significantly more abundant than in those of wild-training I (20.1%) and reintroduced (5.2%) pandas, followed by Leuconostoc(13.0%) and Clostridium (10.9%). Clostridium (40.2%),Leuconostoc (22.8%), and Turicibacter (8.0%) were the most abundant genera in the reintroduced pandas, and were significantly more abundant than in wild-training I (5.5%, 3.8%, and 0.4%, respectively) and wild-training II (10.9%, 13.0%, and 1.7%, respectively) panda gut communities. Clostridium andTuricibacter were notably significantly higher in the reintroduced panda gut communities (40.2% and 8.0%, respectively) compared to those of the wild-training I pandas (10.9% and 1.7%, respectively) to (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4).
The GM composition of reintroduced pandas was more similar to those of wild-training I and wild-training II pandas (Figure 3c). However, significant differences were also observed between the communities of the reintroduced and wild pandas at the phylum and genus levels (p < 0.05, Wilcoxon test) (Supplementary Figure 1). Specifically, Firmicutes (84.2%), Acidobacteria (0.02%), and Cyanobacteria (0.01%) were more abundant in the reintroduced panda communities (p < 0.05, Wilcoxon test) (Supplementary Figure 1a). In contrast, Proteobacteria (51.4%), Bacteroidetes (24.9%), Verrucomicrobia (1.5%), and Actinobacteria (0.8%) were more abundant in wild pandas (p < 0.05, Wilcoxon test). At the genus level, Clostridium (40.2%), Leuconostoc(22.8%), Turicibacter (8.0%), Acinetobacter (3.2%), andYersinia (2.6%) were more prevalent in the reintroduced pandas (p < 0.05, Wilcoxon test) (Supplementary Figure 1b). Conversely, Pseudomonas (28.3%), Sphingobacterium(8.8%), Flavobacterium (6.0%), and Pedobacter (5.4%) were more abundant in the wild pandas (p < 0.05, Wilcoxon test).