3. Results
3.1 Sequence statistics and diversity analysis
A total of 4,723,744 original sequences of the target fragment, with a band size of 415.26 bp, were obtained in 46 fecal samples from wild and captive R. brelichi. Overall, 3,762,171 valid sequences were obtained through primer removal, splicing, mass filtration, deduplication, chimera removal, and clustering of the reads, of which 2,339,533 were from wild R. brelichi and 1,422,638 were from their captive counterparts. The obtained sequences with 100% similarity were merged, and a total of 8,202 ASVs were obtained, with the number of ASVs for each sample ranging from 316 to 734. The rarefaction curves based on the ASVs gradually leveled off with increasing sequencing depth, indicating that most sample information had been obtained from the sequencing data, and no more ASVs were generated with additional sequencing data (Figure 2a). Of the 8,202 ASVs, 427 ASVs were shared by captive and wild R. brelichi , whereas 3,233 and 4,542 ASVs were unique to captive and wild R. brelichi , respectively (Figure 2b).
Alpha diversity analysis showed that the average alpha diversity index of the gut microbial community of captive R. brelichi was higher than that of wild R. brelichi , and there were significant differences in Chao1, ACE, and Shannon indexes between the two groups (p < 0.05). This indicated that the gut microbial diversity and richness of captive R. brelichi were significantly higher than those of wild R. brelichi (Figure 3a). The beta diversity analysis based on the Bray–Curtis distance matrix showed that there were significant differences in gut microbial composition between wild and captive R. brelichi (p < 0.01). In addition, PCoA analysis revealed that wild and captive sample points were significantly separated (Figure 3b). Moreover, NMDS analysis showed that the stress value was 0.09 (less than 0.2), which could better reflect the true arrangement of the data and accurately reflect the extent of difference in the intestinal microbial composition between wild and captive R. brelichi (Figure 3c).
3.2 Gut microbial composition of wild and captive R. brelichi
We identified 8,202 unique ASVs from the 46 fecal samples based on taxonomic annotation, distributed across 28 phyla, 60 classes, 126 orders, 213 families, and 497 genera. At the phylum level, Firmicutes, Bacteroidota, Verrucomicrobiota, Actinobacteriota, Spirochaetota, and Proteobacteria were the core dominant bacterial phyla in all samples, with relative abundances exceeding 1%. Campylobacterota was absent only in samples C14 and C15, and its average relative abundance was 0.23%. Meanwhile, Cyanobacteria was absent only in sample C7, and its average relative abundance was 0.45%. Moreover, Fibrobacterota was absent in samples W1, W5, W12, and C15, with an average relative abundance of 1.87%. Firmicutes, Proteobacteria, and Bacteroidota were the predominant microbes in the gut of wild R. brelichi , accounting for more than 92% of the total relative abundance. Firmicutes, Bacteroidota, Spirochaetota, and Fibrobacterota were the clearly dominant groups of intestinal microorganisms in captive R. brelichi , accounting for more than 94% of the total relative abundance. However, the relative abundance of other bacterial groups was wild R. brelichi was higher than that of their captive counterparts.
At the genus level, the bacteria with ≥1% abundance were selected for analysis, and the bacteria with unclassified abundance or relative abundance of <1% were classified as others. UCG-005 ,Clostridia _UCG-014, Christensenellaceae_R-7 _group,[Eubacterium]_coprostanoligenes_group , Muribaculaceae (unidentified genus), NK4A214_group , Treponema ,UCG-002 , UCG-010 , Monoglobus , RF39 ,Ruminococcus , and Enterorhabdus were bacterial genera that were common to all samples. The composition of gut microbial dominant groups differed between wild and captive R. brelichi . In the gut microbiota of wild R. brelichi , the Christensenellaceae_R-7 _group showed the highest abundance, followed by Acinetobacter and Clostridia_UCG-014 . Meanwhile, UCG-005 was the most dominant genus of captiveR. brelichi , followed by Christensenellaceae_R-7 _group, Muribaculaceae (unidentified genus), and Fibrobacter . Other dominant genera identified in captive individuals includedTreponema and Sphaerochaeta, both belonging to the phylum Spirochaetota. The clustering results indicated that wild and captive individuals clustered into a single group (Figure 4b).
3.3 Analysis of gut microbial differences between wild and captive