Interactions between host sex and seasonal changes shape the gut microbial communities of wild blue sheep (Pseudois nayaur)

Abstract

The construction of gut microbial communities in wildlife is influenced by both environmental factors and host genetic background. However, the mechanisms through which these factors interact to shape microbial communities remain poorly understood. In this study, we systematically sampled fecal specimens from male and female wild blue sheep across different seasons in Helan Mountain Nature Reserve and analyzed them using 16S rRNA gene sequencing. The objective was to investigate seasonal changes and interactions between sex and season on the gut microbial communities of blue sheep. Our results revealed that Firmicutes and Bacteroidetes were the dominant phyla across all groups, and the ten most abundant genera remain stable across both sexes and seasons. Alpha diversity (Chao1, two-way ANOVA, p = 0.001) and Beta diversity (PCoA, Anosim, R = 0.5410, p = 0.001) analyses further confirmed that seasonal and sex-specific interactions significantly shape the microbial community structure. Notably, the gut microbiomes of male and female blue sheep exhibited distinct response patterns to seasonal changes. LEfSe analysis (LDA > 3) identified 20 microbial taxa with significant seasonal differences, some of which showed sex-specific seasonal variation. These findings highlight the critical role of host sex in modulating the adaptation of gut microbial communities to seasonal environmental stresses. This study provides new insights into the ecological and evolutionary mechanisms of wildlife gut microbiomes and offers a scientific basis for sex-based wildlife conservation strategies.

Keywords: 16S rRNA sequencing; gut microbiome; seasonal changes; sex differences; wildlife conservation.

Figure 1

Sex-specific seasonal dynamics of gut microbiota composition and core microbiome in wild blue sheep. (A) Phylum-level taxonomic composition of the gut microbiota across different seasons and sexes, showing the dominance of Firmicutes and Bacteroidetes. (B) Relative abundance of the top 10 most abundant bacterial genera, revealing distinct seasonal and sex-specific patterns in microbial community structure. (C) UpSet plot demonstrating the shared and unique bacterial ASVs among different sex-season combinations. The horizontal bars represent the total number of ASVs in each group, while the vertical bars indicate the size of intersecting sets. The connected dots below show the specific combinations of shared ASVs.

Figure 2

Sex-specific seasonal shifts in gut microbiota diversity reveal distinct ecological strategies between male and female blue sheep. Alpha diversity reveals contrasting seasonal patterns between sexes in the gut microbiota of wild blue sheep. Female seasonal changes in microbial diversity: (A) Chao1, (B) Simpson, and (C) Shannon indices between summer (SF) and winter (WF) samples. Male seasonal changes in the same diversity indices between summer (SM) and winter (WM) samples: (D) Chao1, (E) Simpson, and (F) Shannon.

Figure 3

Multidimensional analysis reveals distinct seasonal and sex specific structuring of gut microbiota communities in wild blue sheep.(A) Principal Coordinates Analysis (PCoA) based on Bray-Curtis distances demonstrates significant clustering of gut microbiota communities by both season and sex; Sex-specific seasonal differences in community structure shown separately for females (B) and males (C), revealing equally strong but distinct patterns of seasonal community shifts between sexes. (D) Redundancy Analysis (RDA) illustrating the relative contributions of sex and seasonal factors in shaping microbial community composition, with arrows indicating the direction and magnitude of environmental variables. (E) Hierarchical clustering analysis combined with relative abundance heatmap of core ASVs across different sex-season groups.

Figure 4

Sex specific seasonal restructuring of gut microbiota reveals distinct taxonomic signatures and adaptation strategies in wild blue sheep. (A) LEfSe analysis identifying season-discriminative taxa across taxonomic levels (left panel), with Kruskal-Wallis test results showing seasonal abundance variations separately for females and males (right panels). Differential abundance analysis of key bacterial genera between seasons, shown separately for females (B) and males (C). Left panels show relative abundances (%), while right panels display the magnitude and direction of seasonal shifts with 95% confidence intervals. Statistical significance is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5

Support Vector Machine (SVM) analysis identifying discriminative ASVs that drive seasonal patterns in female (A) and male (B) blue sheep. Average importance scores (x-axis) represent the contribution of each ASV to the SVM classification model’s performance in distinguishing seasonal patterns. Adjacent heatmaps display the relative abundance patterns of each ASV across seasonal groups.