3.6 Variation in gut fungal communities of giant pandas with different lifestyles
A total of 4,362,547 fungal ITS sequences were obtained from samples in the lifestyle shift experiments. After removal of mitochondria and chloroplast sequences, 4,218,492 ITS sequences were clustered into 7,438 OTUs at the 97% sequence identity threshold. Fungal community richness was significantly higher in the GM of reintroduced and wild pandas compared to those of wild-training I and wild-training II (p< 0.05, ANOVA test) (Figure 3a). The fungal diversity of wild-training I panda communities was significantly lower than in those of other three groups (p < 0.05, ANOVA test) (Figure 3b). PCoA indicated that fungal communities from hosts with the same lifestyle clustered together and separately from others (Figure 3c).
Ascomycota and Basidiomycota were the dominant phyla of fungal communities in the lifestyle shift experiments regardless of lifestyle, although their abundances varied by lifestyle (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4) (Figure 3d, e). Ascomycota had notably higher relative abundances in wild-training I panda gut communities (92.4%), while Basidiomycota were more abundant in wild-training II (33.4%) pandas (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4) (Figure 3e). At the genus level, Candida (83.1%) was the most dominant genus in wild-training I panda communities and were significantly more abundant than in wild-training II (3.2%) and reintroduced (2.8%) pandas, followed by Williopsis (2.7%) and Cryptococcus (0.7%) (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4) (Figure 3e, f). Cryptococcus (12.3%),Shiraia (10.3%), and Cystofilobasidium (8.1%) were the most abundant genera in the wild-training II panda gut communities. Further, the abundances of Cryptococcus (12.3%),Cystofilobasidium (8.1%), Purpureocillium (3.8%), andPenicillium (2.5%) were significantly higher in wild-training II panda communities than in those of wild-training I (0.7%, 0.7%, 0.06%, and 0.09%, respectively) and reintroduced (3.2%, 0.6%, 0.1%, and 1.0%, respectively) pandas (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4). Mrakiella (9.2%),Phoma (8.3%), and Verticillium (4.9%) were the most abundant genera in the reintroduced pandas. The abundances ofMrakiella (9.2%) were significantly higher in the reintroduced panda communities relative to the wild-training I (0.1%) and wild-training II (0.7%) pandas (Non-parametric factorial Kruskal-Wallis sum-rank test, LDA>4).
Although the gut fungal community compositions in the reintroduced and wild pandas were more similar to each other than they were to those of the wild-training I and wild-training II pandas, significant genus level differences were observed between the reintroduced and wild panda communities (p < 0.05, Wilcoxon test). Specifically, 11 genera were significantly different between reintroduced and wild panda communities (excluding unidentified genera), including Candidaand Calycina which were among the 10 most abundant genera (Supplementary Figure 2). Candida (2.8%) were more abundant in reintroduced pandas, while Calycina (13.2%) was more abundant in wild pandas (p < 0.05, Wilcoxon test).