Abstract
In endangered species conservation, fecal samples are a vital non-invasive tool for gut microbiota analysis. Yet, the influence of external exposure time on microbial composition and function remains unclear, constraining data accuracy and reliability. To address this, we investigated the time-gradient effect in the globally endangered forest musk deer (Moschus berezovskii). Using non-invasive sampling under standardized captive conditions, fecal samples were collected at six storage times: (0, 1, 2, 4, 6, 8 days). Gut microbiota composition, diversity, enterotypes, and functional differences were assessed through 16S rRNA gene sequencing on the Illumina MiSeq platform. In total, 147,013 valid ASVs (amplicon sequence variants) were obtained showing significant shifts in microbial composition with storage time. Dominant phyla included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Increasing storage time led to declining α-diversity, reduced community stability, and more unique genera. PCoA (principal coordinates analysis) and NMDS (non-metric multidimensional scaling) indicated progressive separation of experimental groups from control groups, with Anosim and Adonis confirming progressive separation with storage time. Structurally, Firmicutes decreased while Proteobacteria, specifically the Acinetobacter genus, increased with storage time. Community assembly shifted from deterministic to stochastic processes, reflecting stronger environmental disturbance effects. These results demonstrate that the gut microbiota composition, diversity, and ecological functions in forest musk deer feces are highly sensitive to storage time. Thus, preservation duration must be strictly controlled as a critical variable in microbiome studies. This work establishes methodological standards for non-invasive fecal metagenomics in endangered species, providing theoretical insights and practical guidance for improving scientific rigor in conservation-related microbiome research. KEY POINTS: Fecal microbiota diversity and stability decline significantly with longer storage. Firmicutes decrease while Proteobacteria, especially Acinetobacter, increase over time. Storage duration strongly impacts microbiome data, requiring strict sampling control.