1 | INTRODUCTION
Accumulating evidence indicates that the gut microbiome plays an essential role in modulating the host’s whole-body metabolism (Qin et al., 2012; Turnbaugh et al., 2006), immune system (Sylwia Smolinska, 2016; X. Zhang, Chen, Zhao, & Li, 2020) and adaptation to specific diets and extreme environments (Baniel et al., 2021; H. Li et al., 2018; X. Li et al., 2022; Z. Zhang et al., 2016). Therefore, a deeper understanding of host-microbiome interactions is important for understanding host adaptive evolution and health. In recent years, a reference catalogue of gut microbiome genes has been constructed to reduce data redundancy in large-scale gut metagenomic studies to improve the estimation of the diversity and genetic potential of gut microbiome and serve as a basis for metagenome-wide association studies (Xiao et al., 2016; Xiao et al., 2015; Xie et al., 2021). The catalogue of reference genes is a powerful tool to help us gain a comprehensive understanding of the gut microbiome and is also essential for in-depth functional metagenomic analyses such as species/gene profiling, microbial biomarker discovery, and functional annotation (J. Li et al., 2020; Thingholm et al., 2019). To date, many catalogues of reference genes for the gut microbiome have been established in a variety of mammals, including humans (J. Li et al., 2014; Qin et al., 2010), rodents (Degnan et al., 2012) and ruminants (J. Li et al., 2020; Xie et al., 2021). This has greatly promoted knowledge of the interactions between host physiology and their gut microbiome, and is also of great significance for understanding host dietary adaptation and health status monitoring (R. E. Ley et al., 2008; Thingholm et al., 2019).
Nonhuman primates (NHPs) are our closest living relatives and among the most widespread and successful mammals that underwent adaptive radiation. Therefore, studies of the NHP gut microbiome are fundamental in investigations of the ecological and evolutionary adaptation mechanisms of NHPs and even mammals (Amato et al., 2016; Clayton et al., 2018; Goldberg, 2008). Mimicking human dietary patterns in NHP animal models has improved our understanding of the complex interplay between diet, obesity, and the gut microbiome in humans (Newman et al., 2021). Therefore, studies of the NHP gut microbiome may provide important clues for understanding the characteristics of these bacterial communities in the human gut and provide useful guidance and reference for studying the interaction between humans and their gut microbiome (Jianan Sang 2022). In addition, the impact of gut microbiome homeostasis on the conservation of endangered NHPs is of particular concern (Basabose, 2002). Except phylogenetic relationships, the NHP gut microbiome is influenced by multiple other factors, such as diet (Baniel et al., 2021), physiology (Bennett et al., 2016), habitat quality (Barelli et al., 2015), and social interactions (Bennett et al., 2016). For example, the gut microbial structure of geladas (Theropithecus gelada ) adapted to seasonal dietary changes through rapidly change (Baniel et al., 2021). The richness and diversity of gut microbes in the Udzungwa red colobus (Procolobus gordonorum ) were reduced due to dietary changes caused by human disturbance and habitat degradation (Barelli et al., 2015). Therefore, it is very important to study the gut microbiome of NHPs to understand the mechanisms of dietary adaptation and to develop better conservation strategies for these endangered species. However, considering that NHPs occupy a variety of habitats and niches, most previous studies considered a single species or a small number of captive individuals, which is far from being able to completely and accurately represent the general characteristics of intestinal microbiome in NHPs (Degnan et al., 2012; X. Li et al., 2018). The key to detailed analysis of the gut microbiome in NHPs is the availability of comprehensive gene catalogues of wild individuals (Zhu et al., 2020). Therefore, the establishment of a comprehensive reference gene catalogue of the gut microbiome in wild NHPs is of practical significance for further developing the potential resource pool of the gut microbiome in wild NHPs and exploring the relationships of the gut microbiome with ecological adaptation and conservation in NHPs.
Among NHPs, snub-nosed monkeys (Rhinopithecus spp) (SNMs) are a group of endangered colobines including five living species of Sichuan SNM (R. roxellana ), Yunnan SNM (R. bieti ), Guizhou SNM (R. brelichi ), Tonkin SNM (R. avunculus ) and Myanmar SNM (R. strykeri ), of which Yunnan SNMs, endemic to the Tibetan Plateau, are the NHPs with the highest altitudinal distribution (Quan Gouqiang, 2002). Additionally, the living conditions of SNMs are more severe than those of other primates, with features such as low temperatures and limited oxygen (Li BG, 2002; Long YC, 1994), indicating that SNMs should be suitable for studying the mechanisms adaptation to extreme environments. SNMs have sacculated stomachs and are folivores with foregut fermentation. In recent years, the genomic study of SNMs revealed its genetic mechanism underlying dietary adaptations and found evidence for functional evolution in the colobine RNASE1 gene that enables digestion of the high concentrations of bacterial RNA derived from symbiotic microflora (Zhou et al., 2014). Moreover, the digestion of cellulose by the gut microbiome is also an important part of the dietary adaptation of SNMs (Wang et al., 2021). Studies have shown that the gut microbial structure of SNMs is similar to that of ruminants, but the fermentation efficiency is lower than ruminants (D. J. Chivers, Hladik, C.M, 1980). The sacculated stomachs of SNMs do not exhibit the same strong functional division as the rumens of ruminants (D. Chivers, 1994). It is still not fully understood how the gut microbiome and gastrointestinal tract (GIT) help SNMs extract sufficient energy and nutrients from a high-fibre diet (Ganzhorn, 1992; Wang et al., 2021). Previous studies of single SNM species have limited the description of the overall genetic diversity of their gut microbiome (V. L. Hale et al., 2018; V. L. Hale, Tan, C.L., Niu, K., Yang, Y., Zhang, Q., Knight, R., 2019; Yao et al., 2021). Therefore, a comprehensive exploration of the gut microbiome in a large number of wild SNMs is needed to further elucidate their dietary adaptation mechanism, and the establishment of a reference gene catalogue of the gut microbiome in wild SNMs is a reasonable next step. Such a catalogue will provide an important database and framework for analysing the relationships of the gut microbiome with dietary adaptation and for making conservation plans. It will also provide a data basis and reliable criteria for studies of the gut microbiome as well as health, immunity, and adaptation to extreme environments in SNMs.
Here, we constructed an integrated reference gene catalogue of Rhinopithecus gut microbiome (RGC) by processing 143 fecal individual samples from wild populations of SNMs (Yunnan SNM, Sichuan SNM, and Myanmar SNM). The aim of this study was to comprehensively characterize the gut microbiome of SNMs and lay a solid foundation for revealing the relationship between the gut microbiome and host evolutionary adaptation and health. We envisage that the present gut bacterial gene catalogue and functional characterization will serve as a valuable reference and resource for studying the evolution of the gut microbiome in foregut fermenters, also for the study of mammalian evolution and functional adaptation models provides a wide range of perspectives.