The white-headed langur is a highly endangered, karst-endemic primate species with a current wild population of only 1,100 individuals (Konstant et al., 2003). The species inhabits the karst limestone forest in Guangxi, Southwest China, and belongs to the national I class key protected animals (Huang, 2002). White-headed langurs mainly feed on woody plants, which especially focusing on several main plants. Thus, white-headed langurs feed on the tender leaves and buds in the treetops to drink water (Li et al., 2003; Li and Rogers, 2006). In the dry season, white-headed langurs can only feed on old leaves, which contain much less water than new ones. Thus, white-headed langurs adapted to their seasonal habitats by adjusting their ranging behavior and spent more time resting and less time moving and feeding (Huang, 2002; Qihai et al., 2013).

The composition of the gut microbiome was influenced by several factors, including maternal delivery, genetic, geography, and lifestyle (Khan et al., 2016). Diet was an important factor determining the composition of the gut microbiome. Dietary fiber can regulate the contents of Firmicutes and Bacteroidetes, the two main phyla of the human gut microbiome, and increased the abundance of probiotics, such as Lactobacillus, Bifidobacterium, and cellulolytic bacteria (Canfora et al., 2019; Coker et al., 2021). Bacterial abundance was also modulated by dietary macronutrient consumption, including proteins, carbohydrates, and fats (Hildebrandt et al., 2009; Turnbaugh et al., 2009). A previous study found differences in the gut microbiome between captive and wild primates, indicating that captivity humanizes the primate microbiome (Clayton et al., 2016). These data suggest that diet influences the gut microbiome. White-headed langurs mainly feed on leaves, which are coarse fiber and difficult to digest. Whether or not the gut microbiome of white-headed langurs is influenced by their diet is unclear. Over time, the stomachs of white-headed langurs have changed to form a cavity that allows cellulose-breaking bacteria to survive (Zhou et al., 2011). We inferred that the gut microbiome in white-headed langurs is related to their diet.

The karst limestone where white-headed langurs habitated was a harsh environment containing various caves (Huang and Li, 2005). Caves were typical features of a subsurface karst, characterized by darkness, low-to-moderate temperatures, high humidity, and limited nutrients (Gabriel and Northup, 2013). Despite their oligotrophic conditions, microbial communities thrived in caves, with the average number of microorganisms growing in these ecosystems reaching 10⁶ cells/g of rock (Barton and Jurado, 2007). Extremophiles can colonize extreme environments, and they were the sources of novel biomolecules and metabolic pathways (Rothschild and Mancinelli, 2001; Seufferheld et al., 2008). White-headed langurs lived in caves and drunk water in caves when they were thirsty (Huang and Li, 2005). However, whether or not the environment affects the gut microbiome of white-headed langurs was unclear.

In the present study, we collected fecal samples from nine primates, including white-headed langur, François’ langur, silvered langur, loris, pygmy loris, ring-tailed lemur, macaques, gibbon, and baboon, in Guangxi, China. We analyzed and compared the gut microbiome composition and diversity of white-headed langurs and other primates by using high-throughput sequencing. We predicted the functional pathways of the gut microbiome and explored the correlation between environment, diet, and the gut microbiome in white-headed langurs. This study could help improve and protect the living environment of white-headed langurs.

White-headed langurs inhabited the karst limestone forest in Guangxi, Southwest China, fed mainly on leaves, and spend 91.6% of their foraging time on leaves (Huang et al., 2008; Huang et al., 2017). However, how white-headed langurs adapt to the fibrous diet and the karst limestone remains unclear. In the study, result showed that the diversity of the microbiome was higher in white-headed langur than in other primates. The key microbiome in white-headed langur were phylum Firmicutes, class clostridia, family lachnospiraceae, and genera Clostridiates and Ruminococcus, which were related to the digestion and degradation of cellulose. In addition, 10 phylum microbiomes were more enriched in white-headed langur and François’ langur than in other primates, most of which were high-temperature microbiome. The relative abundance of functional pathways involved in glycogen synthesis and degradation in leaf-eating primates were extremely low, indicating the adaptation of white-headed langur to low-energy food.

Change in the gut microbiome was often strongly associated with diet (Barelli et al., 2015; Amato et al., 2016). Food consumption patterns, such as omnivore versus plant-based diet, were associated with changes in the human gut microbiome, which displayed increased abundance of Ruminococcus and Streptococcus, whereas those having a vegetable-based diet show increased abundance of Roseburia, Lachnospira, and Prevotella (De Filippis et al., 2016). Similar to that of humans, the microbiome of non-human primates contains Bacteroidetes, Firmicutes, and Proteobacteria (Yasuda et al., 2015; Nagpal et al., 2018). In our study, Firmicutes, clostridia, lachnospiraceae, Clostridiates and Ruminococcus, which were related to the digestion and degradation of cellulose. The gut microbiome enabled animals to absorb nutrients from the most complex polysaccharides, namely, cellulose and hemicellulose, because the animals themselves did not have the appropriate enzymes in their digestive system (Henderson et al., 2015; Li et al., 2015; Wang et al., 2019). Through degradation and fermentation, these microorganisms decompose plant cellulose into fatty acids and other nutrients to provide their daily energy requirement. Anaerobic and relatively aerobic bacteria (mainly Firmicutes and Bacteroidetes) were the most abundant, along with much smaller quantities of Proteobacteria, Fibrobacteres, Tenericutes, and Actinobacteria (Cholewinska et al., 2020). In the present study, the most abundant phylum in white-headed langur was Firmicutes, followed by Proteobacteria, Actinobacteria, and Bacteroidetes. LEfSe analysis also showed that Firmicutes was the dominant phylum in white-headed langur, and the dominant genera were Clostridiates and Ruminococcus, most of which are related to digestion and degradation. Firmicutes is almost a ubiquitous phylum in nature. Most of its members are spore-forming Gram-positive bacteria and are an essential part of the microbial community associated with plant cellulose degradation and carbohydrate polymer decomposition. Therefore, Firmicutes was important when ligninolytic bacteria and enzymes were desired (Liu et al., 2019). White-headed langurs mainly fed on and drink water from leaves, especially from tender leaves and buds (Huang and Li, 2005; Huang et al., 2008). However, these leaves are coarse cellulose and difficult to digest, and white-headed langurs depend on these bacteria to break down and digest these leaves. These bacteria may be indispensable for the host to adapt to extreme diets or environments. These bacteria were also found in François’ langur and silvered langur, indicating that the gut microbiome is associated with diet.

The microbial composition was mainly influenced by several factors, such as the environment, age, physiological state, diet, and even geographical differences (Henderson et al., 2015; Cholewinska et al., 2020). The karst limestone habitat of white-headed langurs was harsh; it features many cliffs and was covered with various caves (Li and Rogers, 2005). High-temperature limestone in karst is a hotbed of heat-loving bacteria, which could influence the gut microbiome of white-headed langur. In the present study, 10 phyla were significantly higher in white-headed langur than in the other primates, and most members of these phyla were high-temperature bacteria. For example, Abdibacteriota is a Gram-negative, oxygen-demanding, oligotrophic heterodoxygenic bacteria. It can grow under only a limited carbon source and can survive in low-nutrition environments. Phenotypic and genomic analyses have shown that Abdibacteriota was extremely resistant to antibiotics and toxic compounds (Tahon et al., 2018). Aquificae can reproduce in microalkaline sulfide hot springs at temperatures above 70°C and still have a strong nitrogen fixation capacity even under low concentrations of nitrogen compounds; they were considered the main producers of fixed carbon and organic compounds (Nishihara et al., 2018). Caldiserica was a chemical heterotrophic organism that reduced sulfur in hot springs and lived in environments with high temperatures, high salt concentrations, and high pressures, making it difficult to be separated and cultured in the laboratory (Martinez et al., 2019). Candidatus Bipolaricaulota only shown high abundance in white-headed langur. Candidatus Bipolaricaulota was mainly found in deep sea, lake sediments, hot springs, reservoirs, and salt-lake sediments, and it can break down sugar and proteins (Luiken et al., 2019). Calditrichaeota has been recently recognized a novel bacterial phylum with three cultured representatives, isolated from hydrothermal vents, and can degrade detrital proteins (Marshall et al., 2017). Candidatus Saccharibacteria was widely found in soil, sediment, wastewater, and animals, as well as in clinical settings; given its lack of 16S rRNA sequence, little was known about its biological function (Ferrari et al., 2014). Crenarchaeota had low diversity and is thought to be composed of extreme thermophilic bacterial, which was related to system development and metabolism. Thermodesulfobacteria, a small group of thermophilic sulfate-reducing bacteria (Merkel et al., 2017; Guo et al., 2020), can be found in hot springs at high temperatures (>70°C) and was highly abundant in white-headed langur. These high-temperature and cold-resistant bacteria probably originated in karst limestone surface and then transferred to the gut of the white-headed langur. The number of thermophilic bacteria in white-headed langur increased because of the impact of the environment, which helped them adapt to the limestone environment.

We aimed to identify the major function pathway of the gut microbiome in white-headed langurs. Results show that the overall abundance of functional pathways, especially the ribonucleotides biosynthesis related pathways, was lower in white-headed langurs and François’ langurs than in other primates. This result may be related to the energy synthesis because white-headed langur and François’ langur mainly feed on leaves and spend most of their time on rest. Similar to the sugar metabolism pathway, the abundance of sugar metabolism pathways was low in white-headed langur, François’ langur, and silvered langur but high in other omnivorous primates, such as loris, pygmy lorises, ring-tailed lemur, macaque, gibbon, and baboon. This result may be related to their diet because these leaves had less sugar than other food, which led to less sugar decomposition and glycogen synthesis. Thus, the hypometabolic pathway enables the adaptation of white-headed langurs to low-energy food. Ten biological functional pathways unique to white-headed langurs were also found. These pathways are mainly related to the oxidation and degradation of hazardous substances and the biosynthesis of substances. For example, adenine dinucleotide was involved in various physiological activities, such as cell substance metabolism, energy synthesis, and cell DNA repair, which played an important role in the body’s immunity. L-Tryptophan, one of the essential amino acids, was obtained from food. It is an important precursor for 5-serotonin, melatonin, canine uric acid, and niacin. These unique functional pathways could enhance the immunity of white-headed langurs and protect them from the damage of karst limestone.

We analyzed and compared the gut microbiome composition and diversity of white-headed langurs and other primates and explored the influence of diet and karst limestone environment on the gut microbiome of white-headed langur. The gut microbiome in white-headed langur had a high diversity and was associated with diet and karst limestone. The unique functional pathways of white-headed langur were mainly related to the biosynthesis and degradation of harmful substances, enabling them adapting to the karst limestone environment. In addition, the abundance of sugar metabolism-related pathways was low, which was presumably an adaptation to low-energy diet. The results of this study suggest that the gut microbiome is influenced by different factors that help the host adapt to changes in diet and environment.

The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found at Genome Sequence Archive (Genomics, Proteomics, and Bioinformatics 2017) in BIG Data Center (Nucleic Acids Res 2019), Beijing Institute of Genomics (BIG), Chinese Academy of Sciences; https://bigd.big.ac.cn/gsa; CRA002281.

The animal study was reviewed and approved by the Ethics Committee of the Guangxi Chongzuo White-Headed Langur National Nature Reserve Management Center.

TQ and YLH: conceived and designed the project. XP: wrote and edited the manuscript. HH, YW, and YMH: supported and performed laboratory experiments and sequencing. HH and XP: performed the genome assembly and bioinformatics analysis. XP and HH generated the figures and tables. PC, SL, AW, and MH: collected and prepared samples. HL, JW, XR, TQ and YH: review and editing. All authors contributed to the article and approved the submitted version.

This study was funded by the Guangxi Key Research and Development Program (No: Guike AB20059002) and Innovation Project of Guangxi Graduate Education (No: YCBZ2021053). Department of forestry of Guangxi Zhuang Autonmous Region (Guilin Pre-Protection: 2016007).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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