Black-necked cranes are the only species known to inhabit and reproduce on high plateaus throughout their life. The Zoige Wetland National Nature Reserve in China is an important breeding site for black-necked cranes, whereas Caohai and Dashanbao in China are important wintering sites. Black-necked cranes primarily inhabit meadows or marsh meadows in Zoige (Bai et al., 2022), which is one of the hotspots for biodiversity, with plants from the Cyperaceae, Ranunculaceae, and Asteraceae families having the largest number of species. The Zoige area also supports a rich diversity of animal species, including amphibians, fish, and various arthropods such as Diptera and Coleoptera (Xiang et al., 2009). Furthermore, we found arthropods are main animal-deprived food of the black-necked crane’s diet in our previous study (Ma et al., 2024). However, in Caohai and Dashanbao during the wintering period, black-necked cranes primarily inhabit farmland areas (Wu et al., 2013), where they feed on grains, potatoes, and some invertebrates (Dong et al., 2016). We collected black-necked crane feces from 19 locations in Zoige in April and September 2022 as well as in July and November 2023 (spring: N = 30, summer: N = 30, autumn: N = 30, winter: N = 30). The spring and autumn samples are part of a dataset associated with a recently published paper (Ma et al., 2025). During sample collection, we observed black-necked cranes feeding for approximately 2–3 h and collected feces after the birds had left. Using sterile toothpicks, we extracted the internal portion of each fecal sample and placed it into a 15 mL centrifuge tube. The samples were stored in liquid nitrogen and sent to a laboratory in Chengdu, China. We also downloaded the gut microbiome data of black-necked cranes for the wintering period from the National Center of Biotechnology Information (NCBI; project numbers PRJNA681985) (Dashanbao; Zhao et al., 2021), PRJNA992803, and PRJNA995432 (Caohai; Wang et al., 2024). In total, 41 winter samples were obtained from the database (Supplementary Table S1; Figure 1).

Fecal DNA was extracted using an OMEGA Soil DNA Kit (M5635-02; Omega Bio-Tek, Norcross, GA, United States). Negative controls were used for extraction and amplification, and no detectable products were observed. The 16S rRNA region (V3–V4) of the gut microbiota was detected using the primers 338F/806R (Lee et al., 2012). The polymerase chain reaction (PCR; 25 μL) contained: 5 × reaction buffer 5 μL, 5 × GC buffer 5 μL, dNTP (2.5 mM) 2 μL, forward primer (10 uM) 1 μL, reverse primer (10 uM) 1 μL, DNA template 2 μL (20 ng/μL), ddH₂O 8.75 μL, and Q5 DNA polymerase 0.25 μL. The amplification program was as follows: initial denaturation at 98°C for 2 min, denaturation at 98°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s for 30 cycles, and final extension at 72 for °C 5 min. A DNA library was constructed using a TruSeq Nano DNA LT Library Prep Kit (Illumina). Paired-end sequencing of the 16S rRNA gene was conducted using an Illumina NovaSeq 6,000 platform at Personal Bio (Shanghai Personal Biotechnology Co., Ltd., Nanjing, China). The easyAmplicon pipeline¹ was used to process the sequencing data. We used the “fastx_filter” function of VSEARCH (v2.14.1) to trim primers and perform quality filtering. The “derep_fulllength” function of VSEARCH was employed for the dereplication task, with a minimum unique size of 135. To denoise, we used the unoise3 function of USEARCH (v10.0.240), and the “usearch_global” function of VSEARCH was used to generate an amplicon sequence variant table. Rarefaction analysis was conducted using “alpha_div_rare” in USEARCH, and we did not observe obvious batch effects based on cluster dendrogram and PCA analysis (Supplementary Figure S1).

The constrained ordination analysis (CAP) showed that gut microbiota exhibited a seasonal pattern (ANOVA and PERMANOVA, Bray-Curtis: R² = 0.348, p < 0.001, UniFrac: R² = 0.352, p < 0.001; Figures 2A,B), and the unconstrained ordination (NMDS) analysis revealed the same seasonal pattern (ANOVA, Bray-Curtis: R = 0.295 p < 0.001, UniFrac: R = 0.274, p < 0.001) based on Bray-Curtis and weighted UniFrac distances (Supplementary Table S2). They were relatively dispersed across different seasons (Figures 2A,B).

The alpha-diversity during the wintering period was higher than that during the breeding period. There were significant differences among the seasons (Simpson: χ² = 57.719, p < 0.001; Shannon: χ² = 24.494, p < 0.001; Figures 2C,D; Supplementary Table S3). The Simpson indices for the summer and winter Caohai samples were higher than those for the other seasons (Figure 2C; Supplementary Table S4). The Shannon index also indicated that the overwintering Caohai samples had higher diversity than the spring samples (Figure 2D; Supplementary Table S4).

Beta-diversity analysis showed that the wintering period had higher diversity than the breeding period, but the difference was not significant (Figures 2E,F). There were significant differences among the seasons (Bray-Curtis: χ² = 30.907, p < 0.001; UniFrac: χ² = 41.594, p < 0.001).

Summer, CH, and DSB had the highest beta diversity, whereas winter had the lowest diversity based on both Bray-Curtis and UniFrac distance (Figures 2E,F; Supplementary Table S4).

We observed variations in the relative abundances of the gut microbiota. Firmicutes were the dominant phylum in all groups, except for the winter (CH) group in which Proteobacteria were dominant (Supplementary Table S5).

At the family level, Lactobacillaceae were the dominant microbiota (spring: 68.2%; summer: 4.8%; autumn: 54.9%; winter: 76.4%; DSB: 42.2%; CH: 27.3%; Figure 3A; Supplementary Table S6), except for during the summer when Clostridiaceae_1 were dominant (8.7%; Supplementary Table S6). During the summer, certain other microorganisms exhibited relatively high abundances, such as Pseudomonadaceae (summer: 3.0%) and Enterobacteriaceae (summer: 7.2%; Figure 3A; Supplementary Table S6).

We further analyzed seasonal variations in gut microbiota biomarkers. The random forest-based model revealed that the ASV level provided the highest accuracy for classifying gut microbiota across different levels. The cross-validation error rate was 0.13 when using the 36 ASVs identified as having distinct microbiota (Figure 3B). The ASV of Lactobacillaceae was lower in abundance in the summer, whereas the ASVs of Moraxellaceae and Planococcaceae were higher (Figure 3C; Supplementary Figure S3).

In total, 40 nodes (families) and 42 connections (edges) were retained in the black-necked crane co-occurrence network. Only the module did not significantly differ between seasons (Supplementary Table S7). The summer network topology was simpler, whereas the spring and winter exhibited more nodes, edges, and degrees. The nodes, edges, and degrees in winter (CH) and summer were lower than those in other seasons (nodes: χ² = 33.144, p < 0.001, edges: χ² = 36.183, p < 0.001, Figure 4; Supplementary Tables S7, S8).

The βNTI results showed that −2 < βNTI > 2, indicating that the stochastic process is an important factor influencing gut microbiota assembly across all seasons (Figure 5A). ICAMP analysis was performed to evaluate the gut microbiota assembly processes in the different groups. The dispersal limitation (spring: 0.792; summer: 0.612; autumn: 0.728; winter: 0.445; DSB: 0.548; CH: 0.704) was the major driver of gut microbiota assembly in all seasons except winter (Figure 5B; Supplementary Table S9). However, the results for winter contradict the βNTI findings, as homogeneous selection (winter: 0.503), a deterministic process, was the major driver of gut microbiota assembly (Figures 5A,B).

The functional profiles inferred by PICRUSt2 indicated significant variations in the Simpson index (χ² = 13.368, p < 0.05), while the Shannon index did not differ among seasons (χ² = 7.296, p > 0.05). Additionally, both the Simpson and Shannon indices were higher in the summer samples relative to other seasons (Supplementary Figure S4).

Food resources in the environment are crucial factors that influence gut microbiota (diversity and composition) (Scott et al., 2013). In our previous study on the dietary of black-necked cranes in Zoige, we found that a greater diversity of arthropods dietary in black-necked cranes in autumn than in spring (Ma et al., 2024). The current study revealed that a similar trend in the gut microbiota diversity, with the black-necked crane exhibiting lower alpha diversity in spring than in autumn. This suggests that there may be an association between the richness of diet and diversity of gut microbiota. Our findings imply that as dietary diversity increases, so does the diversity of the gut microbiota. Additionally, the current study revealed highest alpha and beta diversity of gut microbiota in summer (Figure 2), which leads us to speculate that the diet of black-necked cranes is most diverse during this period. The abundant water and heat resources in Zoige during summer, which contribute to rich food availability (Zhang Z. et al., 2020), likely support the hypothesis by providing a more varied array of food resources for the cranes. Our findings suggest a link between diet and microbiota diversity, prompting the need for future research on the relationship between dietary intake and gut microbiota composition.

Significant differences in gut microbiota composition have also been reported between seasons of food abundance and scarcity (Orkin et al., 2019). Studies on Tibetan macaques (Macaca thibetana) have shown that during seasons of food abundance, gut microbiota exhibit a higher level of diversity (Sun et al., 2016). To adapt to the abundance of food, the gut microbiota shifts and exhibits higher intra- and inter-species diversity (Zhao et al., 2023). The gut microbiota of the Greater Horseshoe Bats (Rhinolophus ferrumequinum) (Xiao et al., 2019) and Forest Musk Deer (Moschus berezovskii) (Hu X. et al., 2018) were also found to show higher diversity in summer, which has an abundance of food compared with other seasons. Our findings are consistent with those of previous studies. We also observed that the gut microbiota exhibited higher alpha diversity in Caohai and its wintering grounds than at the other locations tested. This could be because human-maintained fields provide ample food, similar to the abundance observed under natural summer conditions (Bergmann et al., 2015).

We found that some gut microbiota families were enriched only in summer (e.g., Moraxellaceae, Planococcaceae, Bacillaceae_1). Moraxellaceae is associated with the benzoate degradation pathway (Torrecillas et al., 2023), Planococcaceae can modulate valine production (Li, 2018; Wu et al., 2024), and Bacillaceae_1 is linked to insect lipids (Li et al., 2022; Weththasinghe et al., 2022). Gut microbiota can rapidly respond to novel food components (Leeming et al., 2019). Previous studies have found that short-term dietary changes alter the gut microbiota of animals; however, these changes are difficult to observe after the return to a normal diet (Leeming et al., 2019). This enrichment likely reflects the animals’ need for diverse materials during digestion.

In summer, black-necked cranes had a more diverse microbiota and a lower relative abundance of dominant bacteria. However, during other seasons, the Lactobacillaceae family was dominant. An increase in a stable gut microbiota may represent an adaptation to cope with harsh environments (Jing et al., 2022; Santos et al., 2024). The Lactobacillaceae family’s strong adaptability allows for long-term colonization, maintenance of the intestinal barrier, and resistance to harmful bacteria, and helps hosts adapt to environmental changes (Santos et al., 2024). Extensive colonization by microorganisms ensures adequate energy intake (Ducarmon et al., 2019). The persistence of colonizing species in animals is likely due to their role in degrading storage carbohydrates, such as starch and fiber (Lee et al., 2024). Lactobacillaceae, known for their involvement in carbohydrate digestion, may colonize the gut for extended periods.

During summer, gut microbial samples revealed high microbial diversity but fewer nodes, edges, and degrees in the co-occurrence network, indicating a simpler network structure. This is likely due to the abundance of available food sources, which enables opportunistic bacteria to thrive and temporarily dominate (Stein et al., 2013). However, transient gut microbiota often have lower competitive adaptability in the gut than long-term colonizing species, which is why they do not persist (Lee et al., 2024). Once the season of food abundance has passed, these transient microbial changes are unlikely to persist. However, this situation is transient, and the complexity of the microbial network is expected to evolve.

We observed more complex microbial networks in other seasons, indicating that the microbiota networks had more nodes, edges, than those in summer. These complexities arise because of the harsh survival challenges that occur outside the summer. High environmental stress may cause the microbiota to establish more positive interactions within communities (Li G. et al., 2019) and support the stress-gradient hypothesis (Bertness and Callaway, 1994; Maestre et al., 2009). To overcome these difficulties, animals adjust their microbial networks to enhance their adaptability by increasing the complexity of their gut microbiota, which can be considered a strategy for biological adaptation to diverse environments (Faust and Raes, 2012). Adaptation has been found in many species, including wild ass (Equus kiang) (Gao et al., 2020), great tit (Parus major) (Bodawatta et al., 2021), Plateau Zokor (Eospalax baileyi) (Liu et al., 2024), and bharal (Pseudois nayaur) (Gao et al., 2024).

Dispersal limitation was the primary driver of the gut microbiota assembly in black-necked cranes during all seasons except winter. Dispersal limitations are important for microbiota assembly. This pattern has been observed in studies on honeybees (Apis cerana and Apis mellifera) (Ge et al., 2021) and birds such as the common nightingale (Luscinia megarhynchos) (Sottas et al., 2021), thrush nightingale (Luscinia luscinia) (Sottas et al., 2021), and green-winged teal (Anas crecca) (Wang et al., 2022). Dispersal limitations reduce the ease with which gut microbes spread between individuals. Previous studies on mammalian gut microbes have found that geographical proximity enhances microbial communication among animals, whereas increased physical distance is a key factor affecting the composition of gut microbes (Moeller et al., 2017). Birds, with higher mobility and broader activity ranges than other animals, experience reduced gut microbiota interactions among individuals (Weinhold, 2022). The reduced interaction of the gut microbiota could be a significant factor affecting the gut microbial composition of black-necked cranes.