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