The animal digestive tract is a complex and dynamic ecosystem with various microorganisms in the digestive tract (Hooper et al., 2001). Studies have shown that bacteria, archaea, and fungal microorganisms are the major groups of intestinal microorganisms (Muegge et al., 2011). Bacteria are the most common intestinal microorganisms (Chen et al., 2018) especially, the phyla that account for a large proportion of the total are Firmicutes, Bacteroidetes, and Proteobacteria (Sommer and Bäckhed, 2013). Numerous studies have proven that intestinal flora is crucial for host survival and reproduction (Hooper et al., 2001; McFall-Ngai et al., 2013). Intestinal flora controls energy homeostasis (Gabanyi et al., 2022), resistance disease and nutrition (Olvera-Rosales et al., 2021) and regulates the immune system (Grizotte-Lake et al., 2018).

Studies have shown that intestinal flora influences many physiological activities such as signaling, immune response, and nutrient metabolism in birds (Kohl, 2012; Grond et al., 2018). Bird gut flora can be influenced by various factors, such as habitat changes (Xie et al., 2016; Wu et al., 2018; Sun et al., 2019), weather changes (Wienemann et al., 2011; Zhang et al., 2020), host genotype (Trevelline et al., 2020; Gao et al., 2021), gender (Cui, 2019), age (Kohl et al., 2019), diet (Xie et al., 2016; Liu et al., 2020a), and health status (Ruiz-Rodríguez et al., 2009; Tong et al., 2018) among others. The dominant gut flora is key in maintaining the normal physiological functions of migratory birds (Grond et al., 2019). However, long-distance migratory birds may carry and transmit microorganisms across geographic areas (Canuti et al., 2019). Trevelline et al. (2019) and West et al. (2019) argue that understanding the microbial communities hosted by individual animals can improve conservation measures for rare wildlife and wild birds. Birds are important ecosystem indicator organisms, and the study of bird-gut microbes is ecologically important. Currently, there are approximately 10,000 bird species worldwide, nearly 2.5 times of mammals, yet research on bird gut microbes is only about 10% of the work on mammalian gut microbes (Grond et al., 2018).

Red-crowned (Grus japonensis) and white-naped (Grus vipio) cranes are large, wading, omnivorous birds. Adult cranes are monogamous and co-inhabit wetland environments. They are considered indicator species for wetland environmental quality (Zou and Wu, 2006; Wu, 2012). In February 2021, both crane species appeared in the National Class I Key Wildlife Protection of China’s National List of Key Wildlife Protection. Moreover, the IUCN Red List (2021) lists both the red-crowned crane and the white-naped crane as vulnerable (VU). The International Crane Foundation (ICF) estimates that 2,800–3,430 red-crowned cranes are in the wild globally. Furthermore, two major populations of red-crowned cranes exist worldwide: island resident populations and continental migratory populations. For white-naped cranes, the worldwide population in the wild is between 6,700–7,700 individuals. The western population winters in the middle and lower reaches of the Yangtze River in China, and the eastern population winters on the Korean Peninsula and Kyushu Island, Japan. Unfortunately, the populations of both crane species are declining in China.

Between March to November, wild red-crowned and white-naped cranes breed in some wetlands of Heilongjiang Province (Zou et al., 2018). The species of plants eaten by the cranes are similar during this breeding period, but the proportions vary. At courtship, the overlap of nutritional and ecological niches between the two populations was 91.67% (Wu, 2012). Although the two crane species are co-dominantly distributed, they differ in their selection and use of the microhabitats (Li, 2017).

Zhalong National Nature Reserve, located in the middle of the East Asia-Australasia bird migration corridor, has the largest reed wetland in Asia (Yang, 2015). The reserve acceded to the Convention on Wetlands of International Importance in 1992 as a waterfowl habitat. Zhalong National Nature Reserve focuses on protecting rare birds and wetland ecosystems, among them are birds including red-crowned and white-naped cranes, the oriental white stork (Ciconia boyciana), Eurasian Spoonbill (Platalea leucorodia), bustard (Otis tarda), and reed parrotbill (Paradoxornis heudei). The Zhalong Reserve is rich in crane resources and is an essential global crane research hotspot. Since the two crane species have similar breeding rhythms and reproductive patterns (Wu, 2012; Hao, 2013; Li, 2017), they can represent the interspecific differences in wild bird intestinal flora.

Therefore, this study used non-invasive sampling methods to collect feces from wild red-crowned and white-naped cranes of the same range. The 16S rRNA high-throughput sequencing technology was applied to analyze the composition and structure of crane intestinal flora. The study hypothesized that: (1) The gut microbial communities of cranes from the same habitats domain have similar composition. (2) The crane intestinal microbial communities perform similar functions. (3) The gut microbial communities of cranes from the same species are more similar than between individuals of different species.

The Zhalong Nature Reserve is among the most important breeding sites for wild and red-crowned cranes in China (Hao, 2013; Li, 2017). For the first time, we used high-throughput sequencing to analyze the intestinal flora of red-crowned and white-naped cranes in the wild. This study annotated 26 phyla and 523 genera in the gut of the red-crowned crane and 25 phyla and 625 genera in the gut of the white-naped cranes, which is significantly greater than previous results on the same species (Trevelline et al., 2020). This difference may be because the cranes in this study were wild and lived in a complex, variable environment. In contrast, the cranes in the Trevelline et al. (2020) study were caged and lived in a more homogeneous environment.

Firmicutes had the highest relative abundance in the gut of both crane species at the phylum level. This observation is consistent with that from the gut of 13 other cranes and many waterfowl species such as egrets (Egretta garzetta), black-crowned night heron (Nycticorax nycticorax), cormorant (Phalacrocorax carbo), and black swans (Cygnus atratus). Numerous studies have proven that Firmicutes are the most abundant in the intestines of almost all vertebrates, not only birds (Ding et al., 2017). This phenomenon may be greatly related to the function of Firmicutes, which degrade sugars and fats (Bäckhed et al., 2004) in intestinal food to produce energy and other nutrients for direct use by the host. Thus, they improve food utilization by the host (Flint et al., 2008). In addition, Firmicutes metabolize carbohydrates to produce short-chain fatty acids (SCFAs), which are also closely linked to host immune regulation (Atarashi et al., 2011; Zhang et al., 2015). Proteobacteria and Bacteroidetes were also common dominant groups in the intestines of both cranes, consistent with the dominant phylum in the intestines of chickens (Gallus gallus domesticus), turkeys (Meleagris gallopavo) (Wei et al., 2013), and 15 cranes species (Trevelline et al., 2020). The gut of swans also has similar dominant phyla composition (Wang et al., 2021). Proteobacteria are a very complex bacterial phylum that utilizes carbon-derived substances and is important in energy accumulation (Lu et al., 2012). Moreover, Proteobacteria have various physiological functions that are important for maintaining the homeostasis of their intestinal microbial communities and the healthy growth and development of their hosts (Tenaillon et al., 2010; Samanta et al., 2012). Bacteroidetes mainly ferment carbohydrates, polysaccharides, proteins, and bile acid, perform steroid metabolism, and promote fat accumulation (Salyers, 1984). This phylum produces butyric acid (Pryde et al., 2002), develops the host immune system, enhances host immunity (Hooper et al., 2001; Hooper, 2004), and are important for balancing the intestinal flora (Sears, 2005). In the spring, both red-crowned and white-naped cranes prefer foods with higher crude protein and crude fat and lower crude fiber (Wu, 2012). Moreover, accumulating Firmicutes, Proteobacteria, and Bacteroidetes benefits both cranes by obtaining sufficient energy and nutrients from these foods. These phyla also facilitate quick recovery of the copious energy consumed during the spring migration and prepare birds for the arrival of the breeding season. Genus Catellicoccus had the highest relative abundance in the gut of both crane species, consistent with the gut of the black-headed gull (Chroicocephalus ridibundus) and the whooper swan (Cygnus cygnus) in the Rongcheng area of Shandong Province (Wang et al., 2021). In contrast, the results differed from those of the cormorant, egret, and black-crowned night heron (Laviad-Shitrit et al., 2019). Catellicoccus is present in most waterfowl and can be used as indicator species for fecal contamination in water or environments contaminated by waterfowl feces (Koskey et al., 2014; Byappanahalli et al., 2015; Safaie et al., 2021). Besides, Catellicoccus also function in nutrient transport and bile acid hydrolysis (Góngora et al., 2021). Lactobacillus was highly abundant in the gut microbial communities of both cranes. Similarly, Lactobacillus was highly abundant in the intestines of other birds, including G. Canadensis (Sandhill Crane) (Trevelline et al., 2020), black-headed gulls (Laviad-Shitrit et al., 2019), black-necked cranes (Grus nigricollis) (Wang et al., 2020), and New Zealand parrots (Strigops habroptilus) (Waite et al., 2012). Moreover, Lactobacillus is a beneficial bacterium in the intestine as it synthesizes vitamins and amino acids required by the animal, promotes mineral absorption and regulates intestinal pH value. Lactobacillus inhibits the growth of pathogenic microorganisms, enhances the barrier function of intestinal epithelial cells, reduces inflammation, and enhances the body’s immune capacity (Artis, 2008; Milani et al., 2017).

In the intestinal flora of the two crane species, a LEfSe analysis revealed Firmicutes as the dominant phylum and Catellicoccus as the dominant genus. The differentially dominant phylum in the intestine of the white-naped crane were Actinobacteria and Bacteroidetes. Normally, the intestinal ratio of Firmicutes to Bacteroidetes (F/B) is related to the strength of fat metabolism in the animal body (Ley et al., 2005). In this study, the intestinal F/B in the red-crowned cranes was higher than that of the white-naped cranes, possibly because the red-crowned cranes have a larger body size compared to the white-naped cranes. Furthermore, the red-crowned cranes create larger nests and lay larger eggs during the breeding season (Hao, 2013; Li, 2017). All these differences normally require more energy consumption. Higher F/B also indicates that the cranes have higher fat metabolism and can obtain higher energy to sustain greater energy expenditure. Additionally, Actinobacteria are a dominant flora in the environment (Wink et al., 2017). The significant abundance of actinobacteria in the gut of the white-naped compared to the red-crowned crane may be due to the close association between the intestinal flora of the white-naped cranes and the environment.

The diversity analysis also showed that the alpha diversity of gut flora was significantly lower in red-crowned than white-naped cranes. Moreover, the Adonis and PCoA analyses showed significantly different structures of the intestinal flora of the two cranes (P < 0.05, R² = 0.139). The intestinal flora diversity of black-necked cranes is significantly different between different regions (Wang et al., 2020), similar to the lesser white-fronted goose (Anser erythropus) (Liu et al., 2020a). During different migration seasons, the gut flora of hooded cranes (Grus monacha) was significantly different (Zhang et al., 2020). We selected wild red-crowned and white-naped cranes from the same distribution area in the spring. Thus, we excluded the influence of spatial and temporal factors on the intestinal flora diversity of the wild cranes. Studies on the lesser white-fronted goose have shown that food habits may influence intestinal flora diversity (Liu et al., 2020a). Wild red-crowned and white-naped cranes in the Zhalong Reserve feed on similar spring food types, and their trophic ecological niche overlap is approximately 90% (Wu, 2012). Therefore, food factors were not considered the main factors influencing the differences in the intestinal flora between the two crane species. Furthermore, Figure 4 showed that white-naped cranes appear to be separated into two clusters, one similar to red-crowned cranes and another separate cluster along the PC1. A possible explanation for this might be that the two species sharing the same foraging ground. According to our observation, white-naped cranes did not migrate to Zhalong wetland at the same time, some of that migrated at different times would forage in groups before the breeding season. Therefore, some individuals of the white-naped crane appear to be clustered and scattered. This result may be explained by the fact that individuals from different small migratory populations arrive at the Zhalong on different dates, while the intestinal flora of the white-naped cranes that arrived later may still retain the environmental adaptation characteristics of some migratory transit sites (Góngora et al., 2021).

By examining the intestinal flora of three species of geese, Yang et al. (2016) suggested that host species may influence the composition of intestinal flora. The gut microbial profile of 59 tropical bird species showed that host species had a greater effect on the gut flora than the host diet and inhabited region (Hird et al., 2015). Both results are consistent with our view that even with closely related species living in the same environment, the gut microbial community structure develops unique characteristics. The top five functional pathways annotated at level 2 contain functional genes that enrich membrane transport, carbohydrate metabolism, amino acid metabolism, and replication and repair. The results generally agree with the predicted gut microbial function of geese and red-crowned cranes in the middle and lower reaches of the Yangtze River (Yang et al., 2016; Dong et al., 2019). Moreover, the results are very similar to those of captive-bred chickens (Zhang, 2021). The predicted gut microbial functions of yak (Bos mutus) and donkey (Equus asinus) are also consistent with the current study (Liu et al., 2019). However, the predicted results of gut microbial functions of the Siberian tiger (Panthera tigris altaica) and tiger frog (Hoplobatrachus rugulosus) differed greatly from those of the crane (Liu et al., 2020b; Ning et al., 2020). Accordingly, we hypothesized that the closer the genetic distance, the more similar the gut microbial function in animals with similar food habits. Besides, the function of gut microbes is related to the adaptation of the host to the external environment (Newell and Douglas, 2014). For example, an enhanced corn (Zea mays L.) diet may promote the gut microbial metabolism of carbohydrates and lipids (Zheng et al., 2019).

In this study, wild red-crowned and white-naped cranes spent a lot of time foraging in the corn field. The high abundance of genes for gut microbial metabolic functions allows both cranes to efficiently obtain numerous nutrients from food to improve their adaptability to food and the environment. The abundance of the main functional groups of microorganisms in the gut of the two cranes was not significantly different (P > 0.05). However, the abundances of the dominant phylum and genera common to the gut of the two crane species were significantly different (P < 0.05). This difference suggests that the gut of different animals with the same domain distribution may be adapted to their respective habits by enriching different species of microorganisms with similar functions.

This study analyzed the gut microbial communities of the red-crowned and white-naped cranes using 16S rRNA high-throughput sequencing. The results showed significant differences in the composition of gut microbial communities between the two crane species in the same range. However, the major functional groups were not significantly different. This study supports the view that host differences are the main reason for the different gut microbial communities. This data will help to develop more scientific conservation plans for cranes.

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 below: https://www.ncbi.nlm.nih.gov/, PRJNA835714.

This animal study was reviewed and approved by Animal Welfare and Animal Ethics Committee of Northeast Forestry University.

ZG, YW, and HZ designed the experiments. ZG, HS, ZL, and YT completed the field sampling. HD, ZG, and XJ performed the data analysis and prepared the figures. ZG wrote the manuscript. YT and ZL contributed to the revision of manuscript. All authors contributed to the article and approved the submitted version.