Gut microbiome and diet contribute to ecological niche differentiation between argali (Ovis ammon hodgsoni) and blue sheep (Pseudois nayaur) on the Qinghai-Tibet Plateau

Abstract

The gut microbiota plays a critical role in plant digestion, nutrient absorption, and ecological adaptation in herbivores. However, how gut microbiota and diet jointly influence ecological niche differentiation in sympatric species remains unclear. Here, we use metagenomic sequencing and plant trnL (UAA) fragment sequencing to examine the gut microbiota and dietary composition of sympatric Tibetan argali (Ovis ammon hodgsoni) and blue sheep (Pseudois nayaur) in the Kunlun Mountains of the Qinghai-Tibet Plateau. Despite inhabiting similar environments, the two species harbor distinct microbial compositions and functional profiles. Interestingly, higher dietary diversity does not correspond to higher microbial diversity. Tibetan argali, despite having a simpler diet, possesses a more diverse and flexible gut microbiome. In contrast, blue sheep show broader dietary preferences and stronger microbial metabolic adaptation to glycan biosynthesis and metabolism. These findings reveal significant associations between gut microbiota composition, function, and diet, supporting a microbial contribution to trophic niche differentiation. Our results highlight distinct microbial-dietary strategies in sympatric herbivores and underscore the role of the gut microbiome in ecological adaptation and species coexistence.

Fig. 1. Overview of gut microbiome composition and gene abundance in Tibetan argali and blue sheep.

a Venn diagram showing shared and unique genes between the Tibetan argali and blue sheep groups. b Bar plot displaying the relative abundance of gut microbiota at the phylum level for each sample. c Box plot comparing the relative abundance ratio of Bacillota to Bacteroidota between groups. d Heatmap illustrating the genus-level relative abundance of gut microbiota across all samples. Samples labeled with “PYA” represent Tibetan argali, and “YYF” represent blue sheep. *: P < 0.05, **: P < 0.01, calculated by Wilcoxon rank-sum test. n = 9 biologically independent samples per group.

Fig. 2. Diversity analysis and LEfSe analysis of the gut microbiota.

a Comparison of α-diversity indices between groups using Welch’s t-test. Boxplots show the median (center line), 25th and 75th percentiles (box bounds), and the full range from minimum to maximum (whiskers). b Non-metric multidimensional scaling (NMDS) plot showing gut microbial community structure. c Histogram of linear discriminant analysis (LDA) scores identifying group-specific microbial biomarkers (LDA score > 4). *: P < 0.05, **: P < 0.01. n = 9 biologically independent samples per group.

Fig. 3. Functional differences in the gut microbiota between Tibetan argali and blue sheep.

a Comparison of KEGG level 1 and level 2 functional categories. Color represents KEGG level 1 categories; corresponding level 2 functions are shown with matching colors. b Heatmap of Spearman correlations between dominant bacterial genera and KEGG level 2 functional categories. c Relative abundance of key metabolic pathways (KEGG Orthologies, KOs) related to ABC transporters between groups. d LEfSe analysis of KEGG pathways between the two groups (LDA Value > 3, P < 0.01). *: P < 0.05, **: P < 0.01, calculated by Wilcoxon rank-sum test. n = 9 biologically independent samples per group.

Fig. 4. Distribution and differences in antibiotic resistance genes among groups.

a Bar plot showing the relative abundance of antibiotic resistance ontology (ARO) terms in individual samples. b Violin plot displaying intergroup differences in the top 10 most abundant AROs between Tibetan argali and blue sheep. Dashed lines indicate group medians. c Redundancy analysis (RDA) illustrating associations between the top 10 most abundant AROs and dominant bacterial phyla. The gene “parY” represents the Streptomyces rishiriensis parY mutant conferring resistance to aminocoumarin. Samples labeled with “PYA” represent Tibetan argali, and “YYF” represent blue sheep. **: P < 0.01, calculated by Wilcoxon rank-sum test. n = 9 biologically independent samples per group.

Fig. 5. Dietary composition and its relationship with gut microbiota diversity.

a Relative abundance of plant families detected in each sample. b Comparison of dietary diversity at the family level using Welch’s t-test. Boxplots show the median (center line), 25th and 75th percentiles (box bounds), and the full range from minimum to maximum (whiskers). c Linear regression analysis between dietary diversity and microbial diversity at the family level. d Random forest analysis showing the impact of dietary composition on microbial diversity in blue sheep. e Random forest analysis showing the impact of dietary composition on microbial diversity in Tibetan argali. Samples labeled with “PYA” represent Tibetan argali, and “YYF” represent blue sheep. *: P < 0.05, **: P < 0.01. n = 9 biologically independent samples per group.

Fig. 6. Correlation and co-occurrence networks linking dietary composition with gut microbial genera and functional profiles.

a Correlation network between dominant genera and dietary composition in Tibetan argali; b Correlation network between dominant genera and dietary composition in blue sheep; c Co-occurrence network of dietary composition and gut microbiome functions in Tibetan argali; d Co-occurrence network of dietary composition and gut microbiome functions in blue sheep. Edges represent significant Spearman correlations; edge thickness reflects correlation strength. *: P < 0.05, **: P < 0.01. n = 9 biologically independent samples per group.

Fig. 7. Sampling map of Tibetan argali and blue sheep in the Kunlun Mountains region of the Qinghai–Tibet Plateau.

The map shows sampling sites of the two species across the study region. The basemap was generated using ArcGIS Pro (Esri, USA). National, provincial, and plateau boundary data were obtained from the Resource and Environment Science and Data Center (RESDC, http://www.resdc.cn). Photographs of Tibetan argali and blue sheep were taken by the authors.