Avian species play an essential ecological function in natural ecosystems, and the study of their gut microbiomes will help us understand birds’ evolutionary biology, contributing to the protection of endangered species and their living environment (Hird, 2017; Grond et al., 2018; Ingala et al., 2018). Numerous studies have shown that habitat conditions and diet could affect the gut microbial community of wild animals (Davidson et al., 2020), and the intestinal microbiome may also change to help the host adapt to different environments (Youngblut et al., 2019). Currently, abundant studies have shown that migratory bird feces can increase the carbon, nitrogen, and phosphorus content in wetland soil, which helps maintain the diversity of wetland vegetation (Irick et al., 2015; Wu et al., 2018).

Previous studies have shown that most of the world’s birds are migratory (Grond et al., 2018), and monitoring the health of migratory birds at stopover, breeding, and wintering sites is important for the conservation of the species concerned, especially at wintering sites, which are directly related to the survival of their populations (Zhao et al., 2021). However, due to human activities and other reasons, the wintering sites of some bird species show a shrinking trend or fragmentation characteristics (Song et al., 2014). Therefore, focusing on the intestinal flora characteristics of migratory birds, especially for the wintering period of rare and endangered birds, has positive implications for the conservation of this species. Studies have illustrated that the diet and living environments directly affect the gut microbiome composition of birds (Jiang et al., 2020; Wang et al., 2021), especially migratory birds whose living conditions change during the different life activity cycles (Zhao et al., 2021). For example, Zhou et al. (2016) found significant differences between the gut microbiome communities of Tibetan chickens from five different habitats (Zhou et al., 2016). Furthermore, Jarma et al. (2021) analyzed the bacterial community of migratory waterbirds and found that the intestinal microbiome of these birds varied according to changes in feeding habits (Jarma et al., 2021). Zhao et al. (2021) elucidated the gut microbiome of black-necked cranes (Grus nigricollis) between early and late wintering periods, understanding the adaption mechanism of migratory birds (Zhao et al., 2021).

The relict gull (Larus relictus), a migratory bird, is classified as “vulnerable” in the International Union for Conservation of Nature Red List (BirdLife International, 2017; Yang et al., 2021; Wang et al., 2022). Their main breeding grounds are the Russian Far East, eastern Mongolia, and Kazakhstan; every winter, the gulls migrate from colder places to spend winter in China (Liu et al., 2017). Most individual wintering sites of the relict gulls are located in the coastal mudflats of Bohai Bay, China (Liu et al., 2017), but Tianjin Relict Gull Park is one of their main wintering sites in Tianjin, China. During migration, wild birds can obtain food from different environments and interact with other birds (Pekarsky et al., 2021). Therefore, their potential to transmit microorganisms is high (Araujo et al., 2014), and monitoring their health levels will not only help conserve endangered migratory birds but also in the timely detection of the transmission of pathogenic microorganisms. Thus, studying microorganisms from fecal samples will provide a tool to monitor potential pathogens (Turjeman et al., 2020), and changes during the overwintering period will lay the foundation for analyzing the adaptation mechanisms of relict gulls to the living environment (Sun et al., 2022). Numerous studies have shown that the dynamic adjustment of the gut microbiome composition allows the host to absorb food more efficiently to meet their energy and nutrient requirements (Hooper et al., 2002; Koren et al., 2012; Chevalier et al., 2015). However, previous studies of relict gulls have only compared gut microbiome composition at different breeding sites in Inner Mongolia (Liu et al., 2022), and comparative data on gut microbiome at early and late wintering stages have not been studied.

In this study, we for the first time identified the composition and structure of gut microbiome of relict gulls in the early and late wintering stages from Tianjin city, China, using 16S rRNA high-throughput sequencing. Furthermore, the effects of the gut microbiome on host function were characterized by PICRUST 2 at each overwintering period (early and late). These findings may provide new insight into the gut microbiome composition of L relictus during the overwintering period and contribute to protecting migratory birds during this time period.

The gut microbiome contributes to host health, and studying gut microorganisms will shed light on the different periods during the life cycle of endangered animals (Waite and Taylor, 2014; Zhang et al., 2021b). To the best of our knowledge, the present study is the first to investigate the gut microbiome of relict gulls during the winter, for which we concluded some important findings.

At the phylum level, the gut microbiome was dominated by Firmicutes and Proteobacteria, which have also been found to predominate among swimming birds (e.g. whooper swans (Cygnus cygnus): Wang et al., 2021; Baer’s pochards (Aythya baeri): Xi et al., 2021) and wading birds (e.g. red-crowned cranes (Grus japonensis): Xie et al., 2016; black-necked cranes (Grus nigricollis): Wang et al., 2020; hooded cranes (Grus monacha): Dong et al., 2019; Gu and Zhou, 2021; Zhang et al., 2021a). Furthermore, it has also been demonstrated that the fecal microbiome of Asian crested ibises (Nipponia nippon) (Zhu et al., 2021) and great tits (Parus major) (Goossens et al., 2022) is dominated by Firmicutes and Proteobacteria. Firmicutes have been found in some avian species to help the host break down complex carbohydrates, polysaccharides, and fats (Turjeman et al., 2020), thus improving the ability of hosts to absorb energy and nutrients from daily food. Proteobacteria have several physiological functions and use high-carbon sources, which help to meet the higher energy and nutritional requirements of the organism (Wang et al., 2021; Xi et al., 2021). It is worth noting that although the dominant phyla of avian species converge, there are species-specific dominant phyla, and these interspecific differences are also reflected in the diverse characteristics of the dominant genera of the species, which are related to the evolutionary status (Hoar et al., 2007), habitat environment (Berlow et al., 2021), habits (Risely et al., 2017), and food habits (Davidson et al., 2020; Zhang et al., 2021a) of the avian species. For instance, within the gull genus, the gut flora of Bradford Beach and Grant Park gulls (Koskey et al., 2014) are dominated by Firmicutes and Proteobacteria, with Catellicoccus as the most abundant genus, which is consistent with the results of this study. Furthermore, our results are overall in line with the dominant phylum found in wintering hooded cranes, but hooded cranes were dominated by Clostridium and Epsilonproteobacteria as the dominant genera (Dong et al., 2019), which is inconsistent with the results of this study. This suggests, in part, that the intestinal flora of species with similar evolutionary statuses converge, while the intestinal flora of species with more different evolutionary statuses vary greatly.

To date, only two related reports have been published on the gut microbiome of relict gulls. One study focused on the wild population in June (Liu et al., 2022), whereas the other focused on the captive population in May (Gao et al., 2021). The top two dominant phyla of these studies were similar to our study. Thus, gut microorganisms belonging to the phyla Firmicutes and Proteobacteria may help the relict gulls adapt to living conditions, a result consistent with previous findings in the breeding period of this species (Liu et al., 2022). At the genus level, the results of this study are consistent with those of Liu et al. (2022), with Catellicoccus as the dominant genus. Catellicoccus, belonging to Firmicutes, is widespread in various bird gut microbiomes (Ryu et al., 2014; Laviad-Shitrit et al., 2019) and is detected predominantly in gull feces (Lu et al., 2008; Koskey et al., 2014). The genome of Catellicoccus marimammalium shows that this bacterium encodes multiple functions (e.g., nutrient transport), suggesting that it may have beneficial effects on its hosts (Koskey et al., 2014). Furthermore, studies on thick-billed murres (Uria lomvia) suggested that Catellicoccus may help these birds to optimize their nutrition. Thus, the high abundance of Catellicoccus in wild relict gulls may be related to limited feeding conditions. The results of Liu et al. (2022) and Gao et al. (2021) showed that Lactobacillus was the co-dominant genus. Although no potential association of Lactobacillus has been found in avian studies, it has been shown that its abundance increases during pregnancy and lactation in humans (Koren et al., 2012). Therefore, we speculate that such differences are likely due to the different life histories of the species related to the sampling time.

Firmicutes produces short-chain fatty acid (SCFAs) that can be directly absorbed by the host’s intestinal wall as an energy source (Yadav et al., 2021). Based on the LEfSe analysis, the relative abundance of Firmicutes was significantly higher in the E group than in the L group. Therefore, a high proportion of Firmicutes may help hosts to digest food and improve energy absorption, facilitating their rapid adaptation to the overwintering environment (Zhang et al., 2021a). Erysipelotrichaceae are part of the phylum Firmicutes. Based on previous research, the levels of Erysipelotrichaceae were associated with the host’s carbohydrate consumption (Cox et al., 2013) and may have the ability to produce SCFAs (Li et al., 2020). Wu et al. (2021) also found that this strain could use plant polysaccharides. The abundance of unclassified_Erysipelotrichaceae belonging to the family ‘ Erysipelotrichaceae ‘ within the phylum Firmicutes was higher in the E group than in the L group, consistent with the predicted metabolic pathways, in which the carbohydrate metabolism was also higher in the E group. We speculate that the content of Firmicutes is high in the early period, while the proportion of Firmicutes decreases in the later period, mainly because the living conditions along the migration route are more diverse and involve many plants in addition to fish. After living in overwintering site Tianjin Relict Gull Park, the main food of relict gull is fish, shellfish and other marine organisms (the information was shown in Supplementary Figures 7 , 8 ). As a result, the proportion of bacteria that could help degrade plant polysaccharides in the E group is higher.

Proteobacteria have a variety of metabolic functions that facilitate the high collective requirements for energy and nutrients (Sun et al., 2022). In this study, the abundance of Proteobacteria was significantly higher in the L group than in the E group, which could be a change made by the relict gulls to adapt to cold weather and scarce food resources. This study also found that the relative abundance of Actinobacteriota was significantly higher in the L group than in the E group, consistent with the findings for overwintering of G. nigricollis, but its potential association with avian species has not been studied. We further found a high relative abundance of Fusobacteriota in this study, consistent with the high relative abundance of Fusobacteriota in some carnivorous birds (Waite and Taylor, 2015); therefore, we hypothesize that the high abundance of Fusobacteriota is related to the predominant consumption of high-protein foods by relict gulls during the wintering period ( Supplementary Figures 7 , 8 ). Cetobacterium, which belongs to Fusobacteriota, is predicted to be involved in lipid metabolism (Zhu et al., 2021), and we observed a higher relative abundance of Cetobacterium in the L group than in the E group, improving the utilization of carbon sources after the temperature decreased in the later wintering period. The results suggest that these bacteria may play important functional roles in the relict gulls, such as food digestion, nutrient absorption, and adaptability to the environment, in agreement with previous findings on other avian species (Wang et al., 2021; Yadav et al., 2021; Sun et al., 2022).

Based on beta diversity using both weighted and unweighted UniFrac distances, the two groups were clustered separately and had significant differences. Our finding suggests that the gut microbial composition of the relict gulls varied at the early and late stages of overwintering, similar to many studies demonstrating that different periods of overwintering could have different influences on the compositional structure of microbial communities (Grond et al., 2019; Wang et al., 2021; Zhao et al., 2021) as it relates to different needs. Risely et al. (2018) found no statistically significant differences in microbial communities between migrants and residents of Calidris shorebirds based on alpha diversity (Risely et al., 2018). Inconsistent with the above finding by Risely et al. (2018), the results of this study showed that alpha diversity increased during the overwintering period and may be related to the food source and the living environment in Tianjin city ( Supplementary Figures 7 , 8 ). Furthermore, through functional predictions, the proportion of sequences related to human diseases (e.g., Figure 5 ) was similar between the two groups, indicating that the pathogenic microorganisms carried by relict gulls did not change with migration and wintering grounds. Thus, our analyses of the gut microbiome of the relict gull suggest that the environment and food source for our study site are suitable for L. relictus to survive the winter, to some extent.

In conclusion, this study demonstrates for the first time the microbial community structure of early and late wintering relict gulls. The significant change of some predominant phyla and genera during early and late overwintering periods could be explained as an adaptation to the habitat to some extent. However, it should be mentioned that the limited sample size from one study site likely influenced the statistical power of the present study. Therefore, in future studies, a larger sample size from more habitats could be considered to further determine the comprehensive characteristics of the gut microbiome across the life history of this endangered species.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

DZ, HW, ZZ and HY conceived and designed the study and wrote the manuscript. HY, ZZ, NW, MW, and QW collected the samples and conducted the experiments. DZ, HW, HY, and ZZ analyzed the data. All authors contributed to the article and approved the submitted version.