Diet Analysis And Gut Microbiota Of Herbivorous Anseriformes In The Middle And Lower Yangtze River Floodplain

Greater white-fronted goose Anser albifrons, bean goose Anser fabalis and swan goose Anser cygnoides are the dominant geese species wintering in the Middle and Lower Yangtze River Floodplain. However, current research is limited by methods. For instance, new methods with higher resolution and greater accuracy are needed to complement or replace traditional diet analysis methods, such as microhistologic observation, which is considered time-intensive and low-resolution. In addition, all of these three species live on Carex and Poaceae to get their energy. For herbivorous ruminants, their gut microbiota plays an important role in assisting food digestion. Nonetheless, little is known about those microbes in guts of geese. Regarding all these aspects, we try to combine molecular ecology, microbiology and bio-informatics to get deeper understanding.1. Reference library of wetland plants in Shengjin Lake National Nature ReserveShengjin Lake is one of the most important wintering sites for waterbirds, with about 5-10 percent of population in the Yangtze River Floodplain. The lake provides great abundance of food for waterbirds. We tried to construct a reference library of potential food resources of goose, with an example of Shengjin Lake. First, trnL gene was selected out of eight candidate genes by analyzing representative sequences of representative species downloaded from GenBank. Then, all the potential plants were sampled and the reference library was constructed, which consisted of 87 specimens, belonging to 25 families,53 genera and 70 species. This work provided valuable foundation for further research about wetland plants and for application of metabarcoding to analyze diet of geese.2. Application of metabarcoding to analyze diet of herbivorous geeseFood is one of the most important factors influencing abundance and distribution of waterbirds, which makes the analysis of food composition (diet) a crucial issue. We applied metabarcoding to analyze diet of herbivorous geese, namely greater white-fronted goose (GWFG) and bean goose (BG). Our results revealed that 73% of sequences belonged to Poaceae for GWFG, while 99% of sequences belonged to Carex for BG. With the same samples, microhistologic observations found 13% Poaceae for BG. Besides, two food items were only recovered with microhistologic observations. The dominant food composition is consistent between these two methods. But the metabarcoding provided higher resolution and more information. The discrepancy may derive from the uneven amplification of PCR and other factors. We concluded that metabarcoding is worthwhile to be introduced into waterbird diet analysis. In the meanwhile, microhistologic observation should still serve as a complementary method.3. The correlation between geese gut microbiota and dietAll of these three species live on Carex and Poaceae to get their energy. Nonetheless, little is known about those microbes in guts of geese. Our results indicated the dominant phyla were Firmicutes (49.70%) and Proteobacteria (23.80%). But significant differences were discovered between the same species in two lakes and among three species in the same lake. In Poyang Lake, bean goose harbored the highest diversity of microbes, with a greater abundance of Proteobacteria (50.26%); while swan goose harbored the lowest diversity, but a greater abundance of Firmicutes (78.99%). For greater white-fronted goose, the Shengjin Lake samples were more diverse and contained more Actinobacteria (26.68%). In Poyang Lake, all species fed on Carex, while in Shengjin Lake, greater white-fronted goose fed on a mixture of Carex and Poaceae. Mantel test revealed significant correlation between microbial community structures and diet composition. In total, we found both host species and diet are potential drivers of geese gut microbiota assemblies. This preliminary investigation provided important reference for further researches to understand functions and interactions of these microbes.4. Interactions and functions of microbiota in guts of herbivorous geeseIn addition to community diversity, interaction diversity and function diversity are also important components of biodiversity. Based on 16S sequences, we used RMT (random matrix theory) model to construct molecular ecology networks for four groups of geese (GWFG-SJL, GWFG-PYL, BG-PYL and SG-PYL). We found obvious differences among these groups in terms of network topology and node characters, which confirmed the diversity of interactions among microbes. Furthermore, we used PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states) to predict functional profiles of those microbes. The results showed they performed similar and conservative functions, most of which were involved in nutrient and energy metabolism. However, these attempts were based on 16S sequences, rather than actual metagenomic sequencing. We hope multi-omics will provide better studies in the future.In summary, our research constructed the first reference library of wetland plants in Shengjin Lake National Nature Reserve. Second, we introduced metabarcoding to analyze diets of geese and proved its reliability. Third, for the first time, we characterized the composition and structures of gut microbiota in three geese species. Fourth, we analyzed the interactions of those microbes and predicted their potential function profiles. During the period, we are motivated by the charm and strength of the combination of diverse disciplines. We are looking forward to more such cooperation in the future to promote understanding and protection of geese, waterbirds and other avian. The most innovative contribution of this paper is a) improving the traditional methods for diet analysis; and b) describing the composition of gut microbiota of herbivorous geese and proved the correlation between diet and gut microbiota.