Microbiota composition
After quality control, 12,060 ASVs were obtained. Rarefaction curves demonstrated that the sample sequencing depth was sufficient to reflect the vast majority of microbiota in each sample (Fig. 1a).
The SWF samples had 26 phyla, 58 classes, 130 orders, 219 families, 396 genera, and 165 species. The dominant phyla were Proteobacteria (49.550%), Firmicutes (37.091%), Bacteroidota (4.903%), and Fusobacteriota (4.811%). The SWS samples had 45 phyla, 75 classes, 174 orders, 201 families, 254 genera, and 30 species, with the dominant phyla being Proteobacteria (46.088%), Bacteroidota (24.205%), Chloroflexi (6.602%), and Desulfobacterota (5.679%). The SWW samples had 26 phyla, 41 classes, 103 orders, 139 families, 173 genera, and 33 species, and the dominant phyla were Proteobacteria (82.500%), Bacteroidota (7.112%), and Actinobacteria (6.293%) (Fig. 1b).
21 phyla, 36 classes, 101 orders, 160 families, 276 genera, and 154 species were identified in the FWF samples. The dominant phyla were Firmicutes (51.098%), Proteobacteria (45.084%), Fusobacteriota (1.739%), and Bacteroidota (1.05%). FWS samples had 36 phyla, 73 classes, 150 orders, 200 families, 294 genera, and 36 species, and the dominant phyla were Proteobacteria (71.414%), Desulfobacteriota (6.071%), and Bacteroidota (5.346%), whereas the FWW samples had 33 phyla, 65 classes, 134 families, 175 families, 255 genera and 5.05 genera, and the dominant phyla were Proteobacteria (52.130%), Actinobacteriota (19.365%), Cyanobacteria (17.110%), and Bacteroidota (5.772%) (Fig. 1b).
At the genus level, the dominant genera in the SWF samples were Escherichia-Shigella (14.448%), Enterococcus (10.064%), Vibrio (7.812%), Catellicoccus (5.794%), Bradyrhizobium (5.301%), Cetobacterium (4.511%), Vibrionimonas (4.167%), Paraclostridium (4.075%), and Peptoclostridium (4.021%). The higher abundance of Phaeodactylibacter (16.420%) and Limibaculum (7.335%) was detected in the SWS samples. The SWW samples included Bradyrhizobium (31.803%) and Rhodanobacter (11.111%) (Fig. 1c).
The dominant genera in the FWF samples were Sporosarcina (18.241%), Citrobacter (11.987%), Acinetobacter (6.201%), and Kurthia (5.725%). Dechloromonas (13.325%) was high in FWS samples, whereas Rhodanobacter (14.367%), Cyanobium PCC-6307 (8.664%), and Bradyrhizobium (7.485%) were dominant in the FWW samples (Fig. 1c).
Microbiota diversity
Analyses of α and β diversity were conducted on the fecal microbiota of little egrets living in different habitats. The differences in Chao1, Shannon, and Pd_faith indices were insignificant (p > 0.05) between the two habitats (Fig. 2a-c). In contrast, the results of the PCoA plot based on Weighted_Unifrac distances demonstrated significant differences in the little egret fecal microbiota between the two habitats (p < 0.01; Fig. 2d).
Next, the microbiota of little egret feces and their habitats were compared to investigate the extent of the influence of the environment. Overall, most differences in α-diversity indices between the microbiota of little egret feces and the environment were significant (p < 0.05), whereas β-diversity was significantly different (p < 0.05) in both habitats (Fig. 2a-d), representing a lesser impact of the environment on the microbiota of little egret feces.
Differences in the β-diversity between the environmental samples from SW and FW regions were insignificant (p > 0.05; Fig. 2d), possibly representing microbial conservatism between the environments.
Differences in microbiota composition
For easy comparison, water and soil from the same habitat (SWW and SWS, FWW and FWS) were combined into environmental samples (SWE and FWE) for Venn diagram analysis. Only 26 shared ASVs between the little egrets and environmental samples, 141 shared ASVs between SWF and FWW, 73 shared ASVs between SWF and SWE, and 102 shared ASVs between FWW and FWE (Fig. 3). Significant differences existed in the dominant microbiota between little egrets in the two regions, as well as between little egrets and the environment.
In terms of contribution of major microbiota differences, FWF microbiota had a significantly greater abundance of Bacillales, Bacilli, Planococcaceae, and Sporosarcina than SWF samples, whereas Alphaproteobacteria, Rhizobiales, and Clostridia were more abundant in SWF samples(Fig. 4).
Analysis of functional differences in microbiota
The microbiota functions in little egret feces and the two environments were similar in the relative abundance of the level 1 pathway–both dominated by metabolism (Fig. 5a). The following functions were in the order of relative abundance: environmental information processing, cellular processes, genetic information processing, human diseases, and organismal systems. In the level 2 pathway, the global and overview maps, cell motility, replication and repair, folding, sorting and degradation, and metabolism of cofactors and vitamins of FWF were significantly higher than SWF (p < 0.05), whereas xenobiotic biodegradation and metabolism, cellular community–prokaryotes, metabolism of terpenoids and polyketides, and biosynthesis of other secondary metabolites of SWF were significantly higher (p < 0.05; Fig. 5b). In the level 3 pathway, FWF samples were significantly higher in metabolic pathways, bacterial chemotaxis, and ribosomes (p < 0.05), whereas SWF samples were higher in quorum sensing, microbial metabolism in diverse environments, ABC transporters, and environments (p < 0.05; Fig. 5c).