Variations in gut bacterial communities between lesser white‐fronted geese wintering at Caizi and Shengjin lakes in China

Abstract The avian gut microbiota plays an important role in shaping the health of its host. However, knowledge of gut bacteria in birds lags behind that of other animals. In this study, we investigated the gut bacterial communities of lesser white‐fronted geese (Anser erythropus) wintering at Shengjin Lake and Caizi Lake, China, using high‐throughput sequencing (Illumina MiSeq). Altogether, 1,053,624 high‐quality sequences and 4,405 operational taxonomic units (OTUs) were acquired from 30 fecal samples (15 per lake). The OTUs represented eight phyla and 17 classes from the Caizi Lake samples and seven phyla and 16 classes from the Shengjin Lake samples. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the dominant phyla. The spatial distance and the Chao1, Simpson, and Shannon indices showed that the alpha diversity differed significantly between the samples from both lakes. The phylogenetic tree and heatmap analyses showed that all the Caizi Lake samples were clustered together and all the Shengjin Lake samples were clustered together. These findings suggest that diet may be an important driver of gut microbial community structure in the birds from each lake, and the obvious differentiation in their gut microbial structures may indicate that the bacteria are highly sensitive to food sources at both lakes.

the middle and lower Yangtze River floodplains. Both lakes, which are designated internationally important wetlands, contain abundant aquatic resources and are important stopover and wintering grounds for many East Asian-Australasian migratory geese (Chen et al., 2011). We studied lesser white-fronted geese from Caizi and Shengjin Lakes during their wintering period to compare their gut microbiota. High-throughput sequencing of the 16S rRNA V3-V4 region and statistical analyses were performed to help describe the bacterial community structure and composition, and to determine whether the gut bacterial compositions exhibit the same patterns between the geese at the two wintering locations.

| Ethical standards
No animals were harmed during this research. All experimental procedures complied with current laws regarding animal welfare and research in China and were specifically approved by the Animal Research Ethics Committee of Anhui Medical University.

| Sample collection
Fecal samples were collected from Caizi Lake and Shengjin Lake, the two main wintering sites for lesser white-fronted geese. Both lakes are river-connected shallow lakes of the middle and lower Yangtze River ( Figure 1). Both are globally important wintering habitats for migratory waterbirds on the East Asian-Australasian Flyway (Cao & Fox, 2009;Fox et al., 2011). Fecal samples were collected at foraging sites. Before the samples were collected, telescopes or binoculars were used to observe the geese and select large groups containing more than 150 birds. To avoid human disturbance and soil contamination, fresh fecal samples were collected immediately after the wild birds had finished foraging and had defecated. All samples were collected from the center of each fecal mass (Dong et al., 2019;Xiang et al., 2019), rapidly placed into sterile 50-ml centrifuge tubes, transported to the laboratory, and stored at −80°C.

| Fecal DNA extraction and avian species determination
DNA was extracted from the fecal samples using the Qiagen QIAamp R DNA Stool Mini Kit following the manufacturer's DNA isolation protocol. The extracted DNA was stored at −80°C. The cytochrome oxidase subunit 1 gene primer pair (BIRDF1: 5′-TTC TCC AAC CAC AAA GAC ATT GGC AC-3′ and BIRDR1: 5′-ACG TGG GAG ATA ATT CCA AAT CCT G-3′) was used for PCR amplification to determine the host species . The cycling conditions were 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1.5 min, and a final extension at 72°C for 10 min. The PCR products were sequenced by Sangon Biotech Co. Ltd., and the resulting sequences were aligned in GenBank (https://www.ncbi.nlm.nih.gov/genba nk/). All samples were confirmed to contain lesser white-fronted goose DNA via sequencing analysis.

| PCR amplification and Illumina MiSeq sequencing
The 338F/806R primer set, equipped with sequencing adapters and unique identifier tags, was used to amplify the bacterial 16S rRNA gene's V3-V4 regions from 30 fecal samples collected from lesser white-fronted geese at Caizi and Shengjin Lakes (15 samples per lake). PCRs were conducted in 50 ml mixtures, each containing 200 mM deoxynucleoside triphosphates, 0.4 mM each of the forward and reverse primers, and 2 U of rTaq DNA polymerase (TaKaRa).
The cycling conditions were 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 60 s, with a final extension at 72°C for 10 min. Tris-boric acid-ethylenediaminetetraacetic acid (2% w/v) agarose gels were used to assess the quality of the amplicons. Amplicons were purified using the MinElute PCR purification kit (Axygen), pooled at equal concentrations, and sequenced to identify the gut bacteria in them using the Illumina MiSeq platform at Oebiotech Co., Ltd. The raw data were submitted to the Sequence Read Archive at the NCBI database (https://www.ncbi.nlm.nih.gov/ sra) under accession numbers SRR9641095-SRR9641124.

| Data analysis
Raw sequencing data were prepared in FASTQ format. Trimmomatic software (version 0.35) was used to preprocess the paired-end reads and detect and excise the ambiguous bases (N). Clean reads were subjected to primer sequence removal and clustered to gen-

| General sequencing information
Thirty fecal samples from lesser white-fronted geese were collected from Caizi Lake and Shengjin Lake, and after processing, the 16S rRNA V3-V4 region gene was sequenced and analyzed. The Illumina MiSeq 2500 sequencing run produced 1,118,001 raw reads. Figure A1 shows the rarefaction curves for each sample. After removing low-quality reads, 1,053,624 clean reads corresponding to 4,405 OTUs were retained. Each sample contained an average of 147 OTUs (range, 93-222 per sample) and 35,120 clean reads (range, 333,655-41,395 per sample). Of the OTUs, 21.77% were found in both populations. Geese from Caizi Lake had 47.98% unique OTUs, and geese from Shengjin Lake had 30.25% unique OTUs ( Figure 2).

| Gut bacterial alpha diversity
Gut bacterial α-diversity was estimated via the observed Chao1, Simpson, and Shannon indices. The spatial distance and the Chao1, Simpson, and Shannon indices showed that the alpha diversity differed significantly between the Caizi Lake and Shengjin Lake samples ( Figure 3). Alpha diversity for the Caizi Lake samples was significantly higher than for the Shengjin Lake samples (p < .01), as indicated by the number of observed OTUs ( Figure 3). Furthermore, the fecal microbiota community compositions differed significantly between the guts of the lesser white-fronted geese from both lakes.
PCA analysis indicated that the samples were well matched with their lakes (Figure 4).
Deferribacteres, Cyanobacteria, Spirochaetae, and Tenericutes accounted for less than 0.001% of all bacteria, a result that was not statistically significant. The dominant class within the Firmicutes phylum was Bacilli (40.92%), Gammaproteobacteria (34.53%) was the dominant class within the Proteobacteria phylum, Actinobacteria (14.54%) was the dominant class within the Actinobacteria phylum, and Bacteroidia (0.85%) was the dominant class within the Bacteroidetes phylum ( Figure A2). Our LEfSe analysis identified differences in the abundances of specific intestinal bacterial taxa in the guts of the lesser white-fronted geese from the two lakes.
Fibrobacteres and Actinobacteria phyla were significantly more F I G U R E 4 PCA of the weighted UniFrac distances for the species sampled from Caizi (CZ) and Shengjin (SJ) Lakes. Red: lesser white-fronted geese from Caizi Lake; blue: lesser white-fronted geese from Shengjin Lake  Bacteroidia abundant in the lesser white-fronted geese from Caizi Lake ( Figure 5), whereas Bacteroidetes and Proteobacteria were significantly more abundant in the geese from Shengjin Lake ( Figure 5).
The phylogenetic tree yielded two major branches: the first branch included the Shengjin Lake samples, and the second branch included the Caizi Lake samples ( Figure 6). The heatmap analysis showed that all 30 samples were clustered into two major groups.
Similar to the phylogenetic tree results, all 15 Caizi Lake samples were clustered together, and all 15 Shengjin Lake samples were clustered together ( Figure A3). Caizi Lake (Figure 7). Almost half of the major functions of the gut bacterial communities in the samples from the lesser whitefronted geese from Caizi Lake were classified into multiple metabolism-related groups (38.15%), including energy production and conversion (5.78%), amino acid transport and metabolism (10.05%), carbohydrate transport and metabolism (8.14%), coenzyme transport and metabolism (4.65%), lipid transport and metabolism (3.99%), inorganic ion transport and metabolism (6.19%), and secondary metabolite biosynthesis (2.38%). There were also 24 KEGG orthologs identified in the gut bacterial communities from the lesser white-fronted geese from Shengjin Lake (Figure 7).

| PICRUSt analysis
Altogether, 42.01% of the major functions of the gut bacterial communities were classified into multiple metabolism groups for the Shengjin Lake samples, including energy production and conversion (5.97%), amino acid transport and metabolism (10.12%), carbohydrate transport and metabolism (6.42%), coenzyme transport and metabolism (4.40%), lipid transport and metabolism (4.31%), inorganic ion transport and metabolism (6.60%), and secondary metabolite biosynthesis (2.81%). The microbial functional classifications appear to be consistent in that most basic metabolic pathways were similar among the individual samples.

F I G U R E 5
LEfSe analysis of the gut bacteria from lesser white-fronted geese at Caizi (CZ) and Shengjin (SJ) Lakes (p < .05) Lesser white-fronted geese are obligate herbivores and long-distance migratory waterbirds found in various ecosystems. However, the distinctive gut bacteria in these birds have received little attention . The present study is the first to explore the gut bacteria from lesser white-fronted geese wintering at Caizi and Shengjin Lakes along the middle and lower reaches of the Yangtze River in eastern China. Our results suggest that a highly diverse gut bacterial community exists in the geese because the geese from the two sampling sites had different taxonomic and ecological bacterial compositions in their fecal samples, and diverse gut bacteria have been reported to display different adaptive mechanisms (Dong et al., 2019). In the present study, the gut bacterial composition and structure in lesser white-fronted geese wintering at Shengjin and Caizi

F I G U R E 6
Lakes were explored. Differences in microbial community structures and interactions were identified. It seems likely that lesser whitefronted geese may modify their digestion to adapt to variations in food availability between Caizi Lake and Shengjin Lake (Wu et al., 2018), and having a diverse gut microbiome may be one such adaptive mechanism.
Here, we found that only 21.77% of all the OTUs were common to geese at both lakes; thus, the community compositions and showed that metabolic pathways accounted for the highest proportion of all the classified bacterial functions, a finding similar to that observed with the gut bacterial community from swan geese in Poyang Lake, China (Wu et al., 2018). Additionally, the bacterial community assemblages showed significant phylogenic clustering, indicating that the gut environment strongly influenced the bacterial community structure. These results suggest that environmental factors can influence the bacterial community composition of lesser white-fronted geese and might be an important factor determining bacterial phylogenetic structuring.
The gut bacterial communities of wintering lesser whitefronted geese may perform many important functions in their hosts. Supporting this assertion, the bacterial community assemblages from our geese showed significant alpha diversity, which indicates that the communities were strongly structured by gut environment filtering (Dong et al., 2019;Yang et al., 2016). The effect of environmental changes on the wintering lesser whitefronted geese relating to bacterial alpha diversity was consistent between Shengjin and Caizi Lakes. Indeed, diet has been found to be an important driver of gut microbial community structure, and the obvious differentiation of the gut bacterial structures may indicate that the gut bacteria were highly sensitive to the food sources available at the two lakes . Caizi and Shengjin Lakes provide lesser white-fronted geese with abundant and diverse food sources while wintering Zhao et al., 2012). However, differences in the diets of the geese at the two lakes were large, with the lesser white-fronted geese feeding almost exclusively on Carex spp. at Caizi Lake, whereas an extra Poaceae spp. component was identified at Shengjin Lake (Wang et al., 2012;Zhao et al., 2012). This suggests that food intake probably influenced the bacterial community compositions in the fecal samples from geese at two lakes and might be an important factor influencing the bacterial phylogenetic structure. The results from the PICRUSt and LEfSe analyses also provide support for diet as a factor influencing the compositions of the bacterial community in the fecal samples from geese at the two lakes. Lesser white-fronted geese feed almost exclusively on Carex spp. at Caizi Lake, and Fibrobacteres and Actinobacteria were significantly more abundant in these geese. Carbohydrate is used as an energy source, and geese can digest simple carbohydrates and complex polysaccharides. Firmicutes and Actinobacteria, which are associated with high levels of carbohydrate metabolism, were also abundant in the Caizi Lake samples (Dong et al., 2019;Xiang et al., 2019). With respect to the Shengjin Lake geese, Bacteroidetes and Proteobacteria, which are associated with Poaceae spp. as food sources (Dong et al., 2019;Xiang et al., 2019), were significantly more abundant in the gut bacterial community from geese at this lake.
Fecal bacteria are often used as biomarkers for studying migratory connectivity in breeding and nonbreeding birds (Møller & Szép, 2011). Between Caizi Lake and Shengjin Lake, the geese in our study group consumed food that differed in type and quality.
The environmental factors were comparatively homogeneous between the two lakes; however, the gut bacteria varied markedly between the geese from each lake. Furthermore, migratory birds also generally show strong-site fidelity for both breeding and wintering locations, often returning to the same location each year during migration and in winter (Møller & Szép, 2011). This may be related to the fact that the lesser white-fronted geese from Caizi Lake had migrated from a different breeding area than those from Shengjin Lake.

ACK N OWLED G M ENTS
This research was supported by the National Natural Science Foundation of China (grant nos. 31702030 and 31700286) awarded to GL.

CO N FLI C T O F I NTE R E S T S
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are provided in full in the results section of this paper, all data was provided by NCBI database ( Caizi Lake Shengjin Lake