Unravelling the metagenomic landscape of gut microbiota in endangered proboscis monkey across Malaysian Borneo: Conservation and ecological insight

Unravelling the metagenomic landscape of gut microbiota in endangered proboscis monkey across Malaysian Borneo: Conservation and ecological insight | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Unravelling the metagenomic landscape of gut microbiota in endangered proboscis monkey across Malaysian Borneo: Conservation and ecological insight Norfarhana Mazlan, Jaya Seelan Sathiya Seelan, Yee Ling Chong, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4487731/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Numerous research on the gut microbial diversity of non-human primates (NHP) has been done to evaluate the impact of environmental changes on the diversity of gut microbiota and to determine the mutualistic relationship between the animals and gut microbes that aid in their adaptation to living in an altered environment. The metagenomics approach was employed to explore gut microbial diversity and abundance in the endangered and endemic proboscis monkeys of Borneo. The amplicon sequencing of the 16S ribosomal RNA (rRNA) gene from 24 faecal samples successfully generated over two million raw reads of bacteria (98%) and archaea (2%) representing 4030 OTUs. Gut microbial diversity and abundance vary across different populations, i.e.: wild, semi-wild and captivity, suggesting that higher diversity was discovered in individuals inhabiting areas with higher food resource availability in natural habitats. The observed alterations in gut microbial diversity and abundance among proboscis monkey populations inhabiting distinct environmental conditions in this study provide empirical evidence for the impact of environmental changes on the proboscis monkey’s gut microbiota. Besides, the prevalence of pathogenic bacteria identified in the proboscis monkey’s gut highlights the potential hazards associated with human-primate interactions and raises the urgent need for public health management. [197 words] Biological sciences/Genetics/Microbial genetics Biological sciences/Ecology Borneo metagenomics microbiome primates Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Non-human primates (NHP) have long been the centre of scientific research, primarily due to their natural behavioural patterns and ecological characteristics that offer insights into the evolutionary processes and adaptations shared with humans. Researchers have been using NHP as a key in comprehending several aspects such as anatomy, behaviour, social structure, physiological and psychological systems that demonstrate significant resemblances to humans [1–4]. The prominent nature of their massive body size which renders them highly conspicuous, along with their extensive geographical range, confer them with a distinct benefit as a study subject. However, unlike humans, the species distribution and habitat selection of non-human primates are highly dependent on their ecological needs which is often correlated with the availability of food resources that becomes the pivotal factor for them to survive [5–8]. Previous studies have demonstrated that the diversity of microbe presence in the monkeys' gut is influenced by the food they consume [9]. This variation has particularly impacted the abundance of beneficial bacteria, which play a crucial role in metabolic processes that facilitate the primates' adaptation to novel sources [9, 10]. Our understanding of the diversity and significance of these microbes in Malaysian primates, particularly in Borneo (Sabah and Sarawak), where 15 primate species reside, remains largely incomplete. Understanding the gut microbiome is essential for unravelling the mutualistic relationship between organisms and gut microbes, especially in light of rapid and extensive habitat changes. This knowledge aids in explaining the adaptation process within changing environments, particularly for endangered species like the proboscis monkey ( Nasalis larvatus ) where habitat loss, degradation, and fragmentation pose significant threats to their survival [11–14]. Proboscis monkeys are large, sexually dimorphic, diurnal arboreal colobines, endemic to the island of Borneo in Southeast Asia [11]. The species has been red-listed by the IUCN as an Endangered species since 2000 [14–15]. They are known to inhabit primarily lowland wetland forests along rivers and coastal areas such as riverine, mangrove, peat swamp and freshwater swamp forests [13–15]. They are folivores, feeding on a variety of plant parts including young leaves or shoots, unripe fruits, including seeds, and flowers [11, 14, 16, 17]. Having a multiple-chambered stomach, the proboscis monkey possesses unique digestive physiology that allows the anaerobic cellulolytic bacteria in their forestomach to ferment their food, and they are capable of regurgitating like the ruminants [16, 18]. Nevertheless, sugar-rich fruits are avoided as the fermentation process will increase acidity in their forestomach and cause bloating that could be fatal to them [14, 19, 20]. In addition, tree bark, termites and termite nest materials are also consumed, though infrequently, by the proboscis monkey for supplement mineral intake, used as a buffer to forestomach pH and removal of toxicity [14]. To date, no information on gut microbiota is available for proboscis monkey populations from Sarawak. Previous research done by Hayakawa et al. [21] focused solely on individuals from Sabah comparing various habitat types such as riverine, mangrove and man-made environment, whereas Jose et al. [22] exclusively described the population from Menanggul River, Sabah. The foremost importance of the information on the gastrointestinal microbiome in proboscis monkeys across geographical regions is for a better interpretation of how different environments and habitat changes influence the diversity of the microbial community of that endangered and endemic species in Borneo. Moreover, the data is useful to assess the health status of the species as the gut microbiome plays an important role in the digestion processes and indirectly benefiting the immune system in many aspects. This can also facilitate the documentation of harmful bacteria present in the gastrointestinal tract of proboscis monkeys, which can be utilized to evaluate the potential of proboscis monkeys as a host reservoir for zoonotic diseases. Therefore, this study was designed to determine the diversity of gut microbiota in proboscis monkeys from Malaysian Borneo and the factors affecting the diversity (e.g., environmental factors and food source availability). It is hypothesised that populations living in natural habitats would possess higher diversity and abundance of gut microbial communities, enabling them to digest a wide range of dietary sources. RESULTS Diversity of proboscis monkeys’ gut microbiota Amplicon sequencing resulted in a total of 2,741,163 paired-end sequencing reads of proboscis monkeys’ 16S ribosomal RNA (rRNA) gut microbes with an average length of 436 base pairs. After the quality filtering processes, a total of 272,086 gut microbiota comprised of 4030 OTUs were discovered from the 24 faecal samples (24 individuals from 7 different localities). Overall, there are 98% of bacteria and 2% of archaea were classified in the analyses. The gastrointestinal bacteria comprised 340 species from 109 families and 20 phyla. Phylum Firmicutes dominated the bacterial phyla that cover 74% of total frequency, followed by Bacteroidetes (15%), and Verrucomicrobia, (3%) (Fig. 1 ). The distribution of the main bacterial phyla identified across seven different localities in Sabah and Sarawak is shown in Fig. 2 . All localities showed a similar pattern with the highest percentage of Firmicutes. Results for all the seven sampling locations from Sarawak and Sabah are given in Table 2 and Fig. 3 . The number of observed OTUs is highest in Bako National Park, Sarawak (Shannon Index, H = 4.015) and lowest in Klias, Sabah (Shannon Index, H = 3.224). Despite the nature of the proboscis population from Lok Kawi which is kept in captivity, they sustain high gut microbial diversity (Shannon Index, H = 4.127). Table 2 The summary statistics of proboscis gut bacteria from seven populations across Sabah and Sarawak. BNP KLS KNM LB LKW LMNP SWS No. of samples analysed 4 3 4 4 3 3 3 No. of families 56 43 61 46 59 50 43 No. of species 187 102 160 142 175 111 107 No. of individuals 53177 38283 32574 44741 21162 36035 42518 Shannon ( H ) 4.015 3.224 3.865 3.766 4.127 3.530 3.453 Simpson’s 0.9676 0.9136 0.9614 0.9602 0.9697 0.9469 0.9278 * LKW: Lok Kawi Wildlife Park; KLS: Padas Damit Forest Reserve, Garama, Klias; LB: Labuk Bay Proboscis Monkey Sanctuary; LMNP: Limbang Wetland National Park; KNM: Kuala Niah Miri; SWS: Samunsam Wildlife Sanctuary; BNP: Bako National Park. To examine the shift in the gut bacterial diversity across different populations (i.e., wild, semi-wild, and captive; refer to Table 1 ), changes in Beta-diversity were compared, using the Weighted and Unweighted Unifrac distances (Fig. 4 ). The results show the segregation of gut microbial communities in different types of environments (Weighted: F = 1.3286, p = 0.177; Unweighted: F = 1.3939, p = 0.049). The heatmap plot demonstrates major differences in the occurrence of the most abundant genera of the gut microbiota found in proboscis monkeys (Fig. 5 ). There was a vast difference in the microbe composition that largely separated Lok Kawi from other populations, which signifies the changes in microbial diversity from captive proboscis monkeys, supported by the Bray-Curtis dissimilarity clustering shown in Fig. 6 . Whereas the statistically significant Kruskal-Wallis H test ( H = 14.70166, p = 0.022) supports the differences in the gut microbial composition of the proboscis monkey from various localities. Table 1 The proboscis monkey populations were observed in seven selected localities within Sarawak and Sabah. Locality Environmental conditions Accessibility Sarawak Bako National Park (BNP) - Mangrove, peat swamp, mixed dipterocarp forest, heath, beach and cliff vegetations. - Wild populations. - Tourist spot. - Very near to a village. - Direct observation is available. Samunsam Wildlife Sanctuary (SWS) - Mangrove and riverine forest. - Wild populations. - Not accessible to public. Kuala Niah, Miri (KNM) - Mangrove forest. - Wild populations. - Near to villages. - Observation only available by boat. Limbang Wetland National Park (LMNP) - Mangrove forest. - Wild populations. - Near to a village. - Observation only available by boat. Sabah Labuk Bay Proboscis Monkey Sanctuary (LB) - Mangrove and mixed dipterocarp forest. - Semi-wild populations. - Tourist spot. - Direct observation is available. Padas Damit Forest Reserve, Garama, Klias (KLS) - Mangrove and riverine forest. - Wild populations. - Accessible to public. - Observation only available by boat. Lok Kawi Wildlife Park (LKW) - Captive. - Accessible to public. - Direct observation is available. The common bacterial taxa that are shared in all types of habitats comprise the fundamental bacterial community involved in the breakdown processes of complex plant materials, e.g.: Ruminococcaceae, Lachnospiraceae, and Muribaculaceae (Fig. 7 ). Table 3 listed the main bacterial taxa found from the seven populations of proboscis monkeys across Malaysian Borneo, dominated by phylum Firmicutes and Bacteroidetes. Firmicutes were characterized by two main orders, namely, Clostridia and Bacilli. Meanwhile, Bacteroidetes showed no changes in all populations with order Bacteroidia becoming the most significant representative. On the other hand, archaea were only represented by two families which are Methanobacteriaceae and Methanomethylophilaceae, the strictly anaerobes microorganisms. They are the significant methane producers through the metabolism of hydrogen and carbon dioxide. Table 3 Comparison between the main bacterial phyla found in proboscis monkeys from seven localities. Locality Main gut bacterial phyla (and class) recorded 1 2 3 4 Bako National Park Firmicutes (Clostridia, Erysipelotrichia, Negativicutes) Bacteroidetes (Bacteroidia) Verrucomicrobia (Verrucomicrobiae) Proteobacteria (Alphaproteobacteria, Gammaproteobacteria) Samunsam Wildlife Sanctuary Firmicutes (Clostridia, Erysipelotrichia) Bacteroidetes (Bacteroidia) Verrucomicrobia (Verrucomicrobiae) Proteobacteria (Alphaproteobacteria) Kuala Niah, Miri Firmicutes (Bacilli, Clostridia, Negativicutes) Bacteroidetes (Bacteroidia, Ignavibacteria) Verrucomicrobia (Verrucomicrobiae) Actinobacteria (Coriobacteriia) Limbang Wetland National Park Firmicutes (Bacilli, Clostridia, Erysipelotrichia) Bacteroidetes (Bacteroidia) Verrucomicrobia (Verrucomicrobiae) Proteobacteria (Alphaproteobacteria, Gammaproteobacteria) Labuk Bay Proboscis Monkey Sanctuary Firmicutes (Clostridia, Negativicutes) Bacteroidetes (Bacteroidia) Proteobacteria (Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria) Verrucomicrobia (Verrucomicrobiae) Padas Damit Forest Reserve, Garama Klias Firmicutes (Clostridia, Erysipelotrichia) Bacteroidetes (Bacteroidia) Verrucomicrobia (Verrucomicrobiae) Cyanobacteria (Melainabacteria) Lok Kawi Wildlife Park Firmicutes (Bacilli, Clostridia, Erysipelotrichia, Negativicutes) Bacteroidetes (Bacteroidia) Proteobacteria (Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria) Verrucomicrobia (Verrucomicrobiae) DISCUSSION The findings of this study showed that Firmicutes bacteria constitute the highest proportion (73.5%) of the bacterial population, which aligns with previous research on Colobine monkeys which have consistently identified Firmicutes as the predominant phylum in the gut microbiota of these primates [23–27]. The findings are also consistent with the research done on proboscis gut bacterial diversity in Sabah which found Firmicutes to be the most common phylum accounting for 16 to 82 percent of the bacterial composition [21–22]. Firmicutes is a group of anaerobic bacteria, primarily characterised by their gram-positive nature. Notably, several species within this group possess the ability to create endospores, enabling their survival in harsh and extreme environments. Interestingly, the lowest Firmicutes percentage recorded by Hayakawa et al. [21] is only 16% of the individuals sampled in the semi-wild population (mangrove habitat, provisioned with artificial diet items). Meanwhile, this study documented consistent Firmicutes composition in all samples (including semi-wild population) which covers more than 60% of bacteria from every population. Even though the diet items supplied (vegetables cultivated for human consumption) for the semi-wild population comprised of high nutrient content, it drives the shift in bacterial diversity to human-like species composition [26, 28]. However, the comparison and interpretation of data must be done with caution as the study by Hayakawa et al. [21] had only one sample representative from the semi-wild population, while this study analysed a total of four samples from similar habitat types. The Firmicutes bacteria in this study were represented by four major classes: Bacilli, Clostridia, Erysipelotrichia and Negativicutes. Nevertheless, class Clostridia dominated the Firmicutes composition covering 72% of the total frequency with family Ruminococcaceae and Lachnospiraceae as the most recorded families from that phylum. These two bacterial families are the bacteria that commonly occur in the mammalian gut microbiota, previously reported to have a high abundance in herbivorous animals and proven to have efficiency in the digestion of complex plant materials [29–31]. This study clarifies the abundance of Ruminococcaceae and Lachnospiraceae in proboscis monkeys, which is consistent with the findings of Jose et al. [22]. Ruminococcaceae and Lachnospiraceae both assist in the degradation of complex polysaccharides into short-chain fatty acids and are capable of breaking down plant-derived substrate which includes cellulose, hemicellulose, and starch [30]. Species from the family Lachnospiraceae ( Roseburia sp., and Coprococcus sp. ) produced high levels of butyrate significant as the energy source for the colon epithelium [32, 33]. On the other hand, the Eubacterium coprostanoligenes species (family Ruminococcaceae) that is found in this study carry an effective function as the cholesterol-reducing anaerobe, by converting cholesterol to coprostanol. The intestine is not able to absorb coprostanol and it will be excreted by the gastrointestinal system [34–36]. Thus, it is proven that the record of bacteria from these two bacterial families would be a good indicator in assessing the health status of proboscis monkey populations. Not only Firmicutes, Bacteroidetes also provide similar purposes in maintaining the health of proboscis monkeys and become part of important gut microflora in animals. This study recorded the family Muribaculaceae as the main representative from phylum Bacteroidetes, which are also commonly found in the guts of homeothermic animals, e.g., birds and mammals [37]. Covering the highest bacteria percentage after Firmicutes, Bacteroidetes is much needed by the host as it is specialized in degrading high molecular weight organic matter [38, 39]. The primary driver of Muribaculaceae occurrence in the proboscis gut is due to its capability to degrade oxalate, complex carbohydrates (plant cell wall glycans), and encrypt elements of an electron transport chain for energy production. Oxalate and glycan are the main components of the plant cell wall that serve as the protection mechanism and support the energy metabolism of plants [40]. The proboscis monkey also benefited the plant glycan as a fundamental component of the protein folding process (glycosylation) for adaptive immune activation [41, 42]. Interestingly, the number of Muribaculaceae bacteria greatly decreased in the Lok Kawi population indicating captive population may have low varieties of food plant intake. The dietary sources of the proboscis monkey population in Lok Kawi solely depend on the food provided in the park. Thus, this finding is evidence of the fact that reduced natural food sources availability may account for the low abundance of gut bacteria observed in the captive population. Frankel et al. [43] also documented a comparable finding in which folivorous NHPs housed in captivity, display alterations in their gut microbiomes when compared to the wild population. These changes have been attributed to the differences in dietary intake amongst NHP populations. Besides that, similar to the findings of this study, Hayakawa et al. [21] in their study of the semi-wild population reported the largest proportion of the genus Prevotella , which belongs to the phylum Bacteroidetes. In addition to the natural food resources present in their habitat, the proboscis monkeys were provided with supplementary food sources, including vegetables (i.e., carrots and long beans). Consequently, this has resulted in a significant prevalence of Prevotella species, which have been previously shown as the dominant members of the gut microbiota in both humans and captive primates [26, 28] The increase in Prevotella abundance signifies a decline in the monkeys' capacity to metabolise dietary fibre, as Prevotella primarily metabolises simple carbohydrates [27]. Other significant bacterial phyla detected in proboscis’ faecal samples are the Verrucomicrobia, Proteobacteria, Cyanobacteria and Actinobacteria with each phylum covering at least 1% of bacteria of the overall microbial diversity. Verrucomicrobia and Proteobacteria are well portrayed in this study even though they are rarely described as microbiome components of non-human primate gut [25]. The occurrence of Verrucomicrobia, Cyanobacteria, and Actinobacteria in the gastrointestinal tract of the proboscis monkey is commonly attributed to the animal's intake of water. Those bacterial communities are highly prevalent in various environmental settings, frequently observed in both soil and water sources [44, 45]. In addition to inhabiting habitats characterised by aquatic environments, the proboscis monkeys were observed to partake of water sources in close proximity to their feeding grounds. This observation potentially elucidates the presence of bacterial communities within the gastrointestinal tract of the proboscis monkeys. Meanwhile, the detection of Proteobacteria was primarily characterised by the prevalence of species commonly linked to diseases in animals and humans. Hence, it demonstrates the capacity of the proboscis monkey to serve as a reservoir for pathogenic microbes, which will be further discussed below. Pathogen infection typically appears as asymptomatic, although it can lead to severe illness upon accumulation within the host organism. Previous research conducted on non-human primates has identified several pathogenic bacterial genera present in their gastrointestinal tract. These include Escherichia, Salmonella, Mycobacterium, Campylobacter, Helicobacter, Pseudomonas, Shigella , and Yersinia [46–50]. Likewise, the proboscis monkey is not exempt from this scenario and may potentially serve as a substantial reservoir for pathogenic microorganisms. This study identified various pathogens in the faecal samples obtained from the proboscis monkeys in different locations across Malaysian Borneo. Among the most significant findings are from the genera Campylobacter , Helicobacter , and Comamonas . Campylobacter and Helicobacter are known to be pathogenic to both humans and animals and suppress the host immune system by toxin secretion [51]. Previously, McKenna et al. [48] recorded a high abundance of Campylobacter bacteria in symptomatic animals compared to healthy individuals. Due to the sensitivity of the bacteria to the stomach's normal production of hydrochloric acid, low levels of exposure may not result in illness. However, the number of bacteria can swiftly increase as a result. Both Campylobacter and Helicobacter are likely prevalent in the proboscis monkey population, as they are abundantly present in all populations across Malaysian Borneo. On the other hand, the presence of Comamonas bacteria solely in the Lok Kawi region adds to the greater abundance of bacteria that are not shared with the semi-wild population. Comamonas is a prevalent pathogenic bacterium that is frequently encountered in many environmental settings and is known to be a causative agent of diseases in humans. The results obtained in the present study are consistent with previous research conducted by Amato et al. [52] on Colobine monkeys where the captive population exhibited a higher abundance of pathogenic bacteria which heightened susceptibility to gut microbial dysbiosis. In addition to bacteria, the occurrence of archaea in the gastrointestinal tracts of humans, apes and ruminant animals has been documented in previous studies [28, 53, 54, 55]. However, there is a lack of comprehensive research on their metabolic roles. The methanogens are often encountered, and a comparable occurrence has been documented in proboscis monkeys, where 2% of the archaea population belongs to the families Methanobacteriaceae and Methanomethylophylaceae. The methanogens were able to endure in an environment devoid of oxygen and generate methane as a by-product through the process of reducing carbon dioxide using hydrogen or methanol. The archaea belonging to the Methanobacteriaceae family are frequently observed in the human gut and are commonly expelled from the body as flatus or by exhalation [54]. With a compartmentalised stomach, the digestive physiology of the proboscis monkey resembles that of ruminants, and it utilises archaea as part of an important microbial community in food fermentation. Hydrogen, as a fermentation intermediate, will not accumulate in the digestive organ and will instead be metabolised by methanogenic archaea, releasing methane as a by-product [56]. Even though archaea comprise a small portion of the proboscis gut microbiota, they play an important role in the complex metabolic pathways of dietary fermentation in the GI tract of the proboscis monkey. CONCLUSION Studies on the gut microbiota of non-human primates have proved the correlation of bacterial diversity and richness with various aspects that influenced the existence of microbes. Food source availability and diet changes had become the utmost important factors that shaped the microbiota diversity and abundance in the gut, besides the environmental components as a source of bacterial diversity [57]. The unique gastrointestinal physiology of Colobine monkeys allows them to consume fibre-rich foods that are hard to digest and are mostly unpalatable to other animals [58]. This survival strategy is made possible by the existence of diverse gut microbiota presence in their GI tract, and hence, becomes a critical aspect in assessing the health status of the monkey. The discussion of this study brings us to an understanding of the effects of habitat changes on gut microbial composition by evaluating data across different geographic locations and environmental conditions. Remarkably, as the first research done on proboscis’ gut microbiome in Sarawak, information from this research provides an overview of the comparison of gut microbe species from across Borneo in comparison with proboscis data reported previously in Sabah [21–22]. The shifts in the bacterial community were well described as being varied in different types of populations (i.e., wild, semi-wild, and captive), and the gut microbiome of the captive population of proboscis monkeys differed from other populations. In addition, the development of the Pan-Borneo Highway Project that traverses protected areas leads to deforestation and poses a significant risk to the proboscis monkey [59]. With the continuous issues of forest degradation and habitat loss, this Colobine monkey with a very specific diet preference might face problems with insufficient dietary supply. Thus, all the information and knowledge gathered from this study would be very beneficial for long-term planning on the conservation management of proboscis monkeys in Borneo. MATERIALS AND METHODS Field sampling, sample collections and preservation Field samplings were carried out at seven study areas within Sarawak and Sabah (Fig. 8 ). Sarawak; Bako National Park (N 01°43.006’, E 110°26.650’), Samunsam Wildlife Sanctuary (N 01°57.159’, E 109°36.432’), Kuala Niah (N 03°55.855’, E 113°41.794’) and Limbang Wetland National Park (N 04°50.777’, E 115°00.104’). Sabah; Labuk Bay Proboscis Monkey Sanctuary (N 05°56.111’, E 117°48.242’), Lok Kawi Wildlife Park (N 05°50.584’, E 116°49.019’) and Padas Damit Forest Reserve, Klias (N 05°24.404’, E 115°33.180’). The data types of proboscis monkey’s habitat (i.e., wild, semi-wild, captive), and details on accessibility to every location were recorded for reference purposes (Table 1 ). Non-invasive sampling technique was used by collecting the faecal samples to avoid any risk of harm to the species. Surveys on primate (to scout for the faeces) were done three times per day; morning (0630–0900), afternoon (1100–1300) and evening (1600–1830). The faecal samples were collected immediately after defecation and stored in Qiagen RNAlater® Stabilization Solution. DNA extraction, amplification, and amplicon sequencing Microbial DNA were extracted using the QIAamp® Fast DNA Stool Mini Kit modified from the established protocol by QIAGEN Company. Extracted DNA products were sent to a private laboratory (Apical Scientific Sdn. Bhd.) for quality measurement. Amplification (PCR) of the V3-V4 region of the bacterial 16S rRNA gene was done using pyrotagged primers. The purity (ng/ul) and concentration (ratio absorbance at 260/280) of the amplicons were checked using Picogreen and Nanodrop. Samples were proceeded straight for library preparation using Illumina 16S Metagenomics Library Prep Kit and their quality and quantity were determined using Agilent TapeStation 4200. All libraries passed the QC measurement (DNA concentration and molarity of qPCR) and were pooled according to protocol by Illumina before sequencing was done using the MiSeq platform at 2x301PE format. Data analyses a. Sequence Quality Filtering, Alignments and OTU Clustering Data processing was done in QIIME (Quantitative Insight Into Microbial Ecology) software v2.1 [60]. Raw sequence reads were quality filtered, demultiplexed and denoised using DADA2 pipeline v1.6 [61], truncating reads shorter than 240 base pairs. Multiple sequence alignment was done using the MAFFT (Multiple Alignment using Fast Fourier Transform) program and a phylogenetic tree was inferred in FastTree v2.1.10. Sequences were then assigned to its Operational Taxonomic Units (OTUs) with a 95% similarity threshold against the bacterial database available in SILVA [62]. b. Microbial Species Diversity (Alpha and Beta Diversity) To analyze the diversity of microbes between different groups, differential abundance testing was carried out by ANCOM (Analysis of Composition of Microbiome) method [63]. The alpha-diversity (within sample richness) and beta-diversity (between sample dissimilarity) were computed in R package v3.4.1 implemented in QIIME software, as well as PAST (Paleontological Statistics) software v3.0. For the alpha diversity, Shannon ( H ) and Simpson’s Diversity Indices were used, while species evenness was calculated using Buzas-Gibson’s (E) Index. To reveal the factors influencing the diversity of the microbial community, Weighted UniFrac (quantitative) and Unweighted UniFrac (qualitative) distances were tested. The within-group and between-group variance were calculated using Permutational Multivariate Analysis of Variance (PERMANOVA) statistics in an R package with 999 permutations. The Bray-Curtis dissimilarity method and Kruskal-Wallis H test were also applied to observe the changes in gut bacterial diversity between proboscis monkey populations from different types of habitats. Declarations ADDITIONAL INFORMATION The authors declare no competing interest. FUNDING This research has been funded by the Ministry of Higher Education (MoHE) Malaysia under grants NRGS/1087/2013(01) and GL/07/UMS/03/2017 awarded to Associate Prof. Dr Faisal Ali Anwarali Khan and Dr Jaya Seelan Sathiya Seelan. Professor Dr Henry Bernard and research assistant was funded by research grant no. GKP0012-STWN-206 for fieldwork and data collection in Padas Damit Forest Reserve in Klias, Sabah. Author Contribution NM and FAAK contributed as the main researchers. HB, WNSWA, SS and MKZ support the team with fieldworks and sample collections. NM, FAAK, JSS, YLC and HB prepared the manuscript. All authors have read and agreed to the final version of the manuscript. Acknowledgement Permit to conduct sampling activities were approved by Sarawak Forest Department (SFD) (Permit No. NCCD.907.4.4(JLD.13)-265) and Sabah Biodiversity Centre (SaBC) (Permit No. JKM/MBS.1000-2/2 JLD.7 (130)). Thank you to Molecular Genetics lab members of Universiti Malaysia Sabah for providing equipment to conduct lab works in Sabah. Deepest appreciation to all Sarawak Forestry Corporation (SFC) staffs and UNIMAS Primates Research Team for their endless helps during the sampling trip. Finally, we would like to express gratitude to the researchers at Queen Mary University of London (QMUL) for their guidance in conducting the bioinformatics analysis. 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Plant Sci , 274 , 70–79 (2018). Frankel, J. S., Mallott, E. K., Hopper, L. M., Ross, S. R., & Amato, K. R. The effect of captivity on the primate gut microbiome varies with host dietary niche. Am J Primatol , 81(12) , e23061 (2019). Lee, K. C., et al. Phylum Verrucomicrobia representatives share a compartmentalized cell plan with members of bacterial phylum Planctomycetes. BMC Microbiol , 9(1) , 5–14 (2009). Barka, E. A. et al. Taxonomy, physiology, and natural products of Actinobacteria. Microbiol Mol Biol Rev , 80(1) , 1–43 (2016). Anderson, K. F., Kiehlbauch, J., Anderson, D., McClure, H., & Wachsmuth, I. Arcobacter ( Campylobacter ) butzleri -Associated diarrheal illness in a non-human primate population. Infect Immun , 61(5) , 2220–2223 (1993). Sestak, K., et al. Infectious agent, and immune response characteristics of chronic enterocolitis in captive rhesus macaques. Infection , 71(7) , 4079–4086 (2003). McKenna, P., et al. The macaque gut microbiome in health, lentiviral infection, and chronic enterocolitis. PLoS Pathog , 4(2) , e20 (2008). Sasseville, V. G., & Mansfield, K. G. Overview of known non-human primate pathogens with potential to affect colonies used for toxicity testing. J Immunotoxicol , 7(2) , 79–92 (2010). Ghodbane, R., & Drancourt, M. (2013). Non-human sources of Mycobacterium tuberculosis . J Tuberc , 93(6) , 589–595 (2013). Man, S. M. The clinical importance of emerging Campylobacter species. Nat Rev Gastroenterol Hepatol , 8(12) , 669–685 (2011). Amato, K. R. et al. the gut microbiota as a novel tool for examining colobine primate GI health. Glob Ecol Conserv , 7 , 225–237 (2016). Janssen, P. H., & Kirs, M. Structure of the archaeal community of the rumen. Appl Environ Microbiol , 74(12) , 3619–3625 (2008). Gaci, N., Borrel, G., Tottey, W., O’Toole, P. W., & Brugère, J. F. Archaea and the human gut: New beginning of an old story. World J Gastroenterol , 20(43) , 16062 (2014). Raymann, K., Moeller, H. A., Goodman, A. L., & Ochman, H. Unexplored archaeal diversity in the great ape microbiome. ASM , 2(1) , 10.1128/msphere.00026 − 17 (2017). Hungate, R. E. Hydrogen as an intermediate in the rumen fermentation. Arch Microbiol , 59(1–3) , 158–164 (1967). McKenzie, V. J., et al. The effects of captivity on the mammalian gut microbiome. Integr Comp Biol , 57(4) , 690–704 (2017). Amato, K. R. et al. Habitat degradation impacts black howler monkey ( Alouatta pigra ) gastrointestinal microbiomes. ISME J , 7(7) , 1344 (2013). Alamgir, M., Campbell, M. J., Sloan, S., Engert, J., Word, J., Laurance, W. F. Emerging challenges for sustainable development and forest conservation in Sarawak, Borneo. PLOS ONE , 15(3) , e0229614 (2020). Bolyen, E. et al. Reproducible, interactive, scalable, and extensible microbiome data science using QIIME2. Nat Biotechnol , 37(8) , 852–857 (2019). Callahan, B. J., McMurdie, P. J., Rosen, M. J., Han, A. W., Johnson, A. J. A., & Holmes, S. P. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods , 13(7) , 581–583 (2016). Quast, C., et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res , 41(D1) , 590–596 (2012). Mandal., S., Van-Treuren, W., white, R. A., Eggesbo, M., Knight, R., & Peddada, S. D. Analysis of composition of microbiomes: A novel method for studying microbial composition. Microb Ecol Health Dis , 26(1) , 27663 (2015). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4487731","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":313794948,"identity":"56af23cb-89c5-4b0d-aaf4-63a8a62afea7","order_by":0,"name":"Norfarhana Mazlan","email":"","orcid":"","institution":"Universiti Malaysia Sarawak","correspondingAuthor":false,"prefix":"","firstName":"Norfarhana","middleName":"","lastName":"Mazlan","suffix":""},{"id":313794949,"identity":"6d85f615-e3aa-403a-9f4f-8b9207539d50","order_by":1,"name":"Jaya Seelan Sathiya Seelan","email":"","orcid":"","institution":"Universiti Malaysia Sabah","correspondingAuthor":false,"prefix":"","firstName":"Jaya","middleName":"Seelan Sathiya","lastName":"Seelan","suffix":""},{"id":313794950,"identity":"d067e93a-96ad-4955-849f-64e126bdf641","order_by":2,"name":"Yee Ling Chong","email":"","orcid":"","institution":"Education University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Yee","middleName":"Ling","lastName":"Chong","suffix":""},{"id":313794954,"identity":"27082a00-96f5-46cc-94c7-2e7d1bb1be8d","order_by":3,"name":"Henry Bernard","email":"","orcid":"","institution":"Universiti Malaysia Sabah","correspondingAuthor":false,"prefix":"","firstName":"Henry","middleName":"","lastName":"Bernard","suffix":""},{"id":313794956,"identity":"167cb239-d95d-400f-94db-cfdd00741ba0","order_by":4,"name":"Wan Nur Syafinaz Wan Azman","email":"","orcid":"","institution":"Universiti Malaysia Sarawak","correspondingAuthor":false,"prefix":"","firstName":"Wan","middleName":"Nur Syafinaz Wan","lastName":"Azman","suffix":""},{"id":313794957,"identity":"67a6a2e4-5ad9-4565-8d54-eef1e54f7e99","order_by":5,"name":"Sundai Silang","email":"","orcid":"","institution":"Sarawak Forestry Corporation","correspondingAuthor":false,"prefix":"","firstName":"Sundai","middleName":"","lastName":"Silang","suffix":""},{"id":313794958,"identity":"4e37d575-245b-4cf9-94f7-3d77da3a8b1a","order_by":6,"name":"Mohamad Kasyfullah Zaini","email":"","orcid":"","institution":"Sarawak Forestry Corporation","correspondingAuthor":false,"prefix":"","firstName":"Mohamad","middleName":"Kasyfullah","lastName":"Zaini","suffix":""},{"id":313794959,"identity":"d86bb8a9-2a0d-41e3-9a0b-9293d4d8ee8d","order_by":7,"name":"Faisal Ali Anwarali Khan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvUlEQVRIiWNgGAWjYDACHsYHDA8MbBgYmInXwmzAkGCQRrIWhsMkuIu/5zDjh4SC84nb2bkTP/5g2JbYQEiLxNlmZokEg9uJO5t5N0vzMNwmrIXhPP8BsJYNh3k3SDMQo0X+PDPzjwSDcyAtm3/+IEaLwdlmNqAtB0BatkkQ5TDDM4fZLBIMko1BWqx5DG4bE9QidyaZ+caHP3ayG86f3XzzR8VtWYJa0N3J4EiqFgYGe5J1jIJRMApGwbAHAJfwQTUMaQYtAAAAAElFTkSuQmCC","orcid":"","institution":"Universiti Malaysia Sarawak","correspondingAuthor":true,"prefix":"","firstName":"Faisal","middleName":"Ali Anwarali","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2024-05-28 03:03:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4487731/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4487731/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59137703,"identity":"b8b7c947-8e28-469c-8bc4-b4301859fef2","added_by":"auto","created_at":"2024-06-26 19:00:37","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":356275,"visible":true,"origin":"","legend":"\u003cp\u003eThe gut bacteria composition isolated from proboscis monkey faecal samples in Malaysian Borneo. The bacterial phyla with less than 1% composition were categorised as others.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/975a16445e5beff05d632e4c.jpeg"},{"id":59137698,"identity":"67c6d7ce-fc44-4027-b8e1-d2f1886308cc","added_by":"auto","created_at":"2024-06-26 19:00:36","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":276626,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative abundance of proboscis’ gut bacterial phyla from seven localities in Sarawak and Sabah. \u003cstrong\u003e*\u003c/strong\u003eLKW: Lok Kawi Wildlife Park; KLS: Padas Damit Forest Reserve, Garama, Klias; LB: Labuk Bay Proboscis Monkey Sanctuary; LMNP: Limbang Wetland National Park; KNM: Kuala Niah Miri; SWS: Samunsam Wildlife Sanctuary; BNP: Bako National Park.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/5ec86b496983dd01ab32c028.jpeg"},{"id":59137708,"identity":"485f9460-6fac-4b55-869b-9af6c49a06c7","added_by":"auto","created_at":"2024-06-26 19:00:37","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118678,"visible":true,"origin":"","legend":"\u003cp\u003eSpecies richness index of gut bacteria isolated from proboscis’s faecal samples. \u003cstrong\u003e*\u003c/strong\u003eLKW: Lok Kawi Wildlife Park; KLS: Padas Damit Forest Reserve, Garama, Klias; LB: Labuk Bay Proboscis Monkey Sanctuary; LMNP: Limbang Wetland National Park; KNM: Kuala Niah Miri; SWS: Samunsam Wildlife Sanctuary; BNP: Bako National Park.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/4a77e176533b844cc32b7e97.jpeg"},{"id":59137709,"identity":"8dcaec8f-6097-407a-b4bf-74a8265bb1a1","added_by":"auto","created_at":"2024-06-26 19:00:37","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":197504,"visible":true,"origin":"","legend":"\u003cp\u003eThe PCoA plots using (A) Weighted Unifrac and (B) Unweighted Unifrac distances show changes in gastrointestinal microbial community composition comparing different habitats of proboscis monkeys.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/720ffd3ff29b516beb29679b.jpeg"},{"id":59137710,"identity":"e7944ea0-3ef1-465f-818c-fd2f56280cfa","added_by":"auto","created_at":"2024-06-26 19:00:37","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1024650,"visible":true,"origin":"","legend":"\u003cp\u003eThe heatmap plot demonstrates major differences in the occurrence of the common bacterial families found in proboscis monkeys from seven different localities.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/4c4019e7ff34baaa078b1387.jpeg"},{"id":59137701,"identity":"fa80231c-3989-4573-a3e5-37d53cd56488","added_by":"auto","created_at":"2024-06-26 19:00:36","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":105177,"visible":true,"origin":"","legend":"\u003cp\u003eBray-Curtis dissimilarity clustering of the changes in gut microbiota of proboscis monkeys across seven different locations.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/46d2d1eadac4d74fc2eb3ca0.jpeg"},{"id":59137707,"identity":"3fc182e9-020b-400e-8555-cc7a096c6f59","added_by":"auto","created_at":"2024-06-26 19:00:37","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":85772,"visible":true,"origin":"","legend":"\u003cp\u003eVenn diagram illustrates the bacterial OTUs found in the gastrointestinal tract of proboscis monkey sampled from three different types of habitats.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/708d12294b671d758b4d144b.jpeg"},{"id":59137702,"identity":"4519cd01-bc9b-4d4a-84bf-a0934acd3e67","added_by":"auto","created_at":"2024-06-26 19:00:36","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":270675,"visible":true,"origin":"","legend":"\u003cp\u003eSelected sampling locations for this study throughout Sarawak and Sabah.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/dfed4a42c6814a85774884ea.jpeg"},{"id":62328431,"identity":"2fbdc69f-4256-4a8e-ada0-57ae06eb9186","added_by":"auto","created_at":"2024-08-13 03:15:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3146936,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4487731/v1/e098d583-2907-4663-9c13-8d599f2e7d25.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Unravelling the metagenomic landscape of gut microbiota in endangered proboscis monkey across Malaysian Borneo: Conservation and ecological insight","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNon-human primates (NHP) have long been the centre of scientific research, primarily due to their natural behavioural patterns and ecological characteristics that offer insights into the evolutionary processes and adaptations shared with humans. Researchers have been using NHP as a key in comprehending several aspects such as anatomy, behaviour, social structure, physiological and psychological systems that demonstrate significant resemblances to humans [1\u0026ndash;4]. The prominent nature of their massive body size which renders them highly conspicuous, along with their extensive geographical range, confer them with a distinct benefit as a study subject.\u003c/p\u003e \u003cp\u003eHowever, unlike humans, the species distribution and habitat selection of non-human primates are highly dependent on their ecological needs which is often correlated with the availability of food resources that becomes the pivotal factor for them to survive [5\u0026ndash;8]. Previous studies have demonstrated that the diversity of microbe presence in the monkeys' gut is influenced by the food they consume [9]. This variation has particularly impacted the abundance of beneficial bacteria, which play a crucial role in metabolic processes that facilitate the primates' adaptation to novel sources [9, 10]. Our understanding of the diversity and significance of these microbes in Malaysian primates, particularly in Borneo (Sabah and Sarawak), where 15 primate species reside, remains largely incomplete. Understanding the gut microbiome is essential for unravelling the mutualistic relationship between organisms and gut microbes, especially in light of rapid and extensive habitat changes. This knowledge aids in explaining the adaptation process within changing environments, particularly for endangered species like the proboscis monkey (\u003cem\u003eNasalis larvatus\u003c/em\u003e) where habitat loss, degradation, and fragmentation pose significant threats to their survival [11\u0026ndash;14].\u003c/p\u003e \u003cp\u003eProboscis monkeys are large, sexually dimorphic, diurnal arboreal colobines, endemic to the island of Borneo in Southeast Asia [11]. The species has been red-listed by the IUCN as an Endangered species since 2000 [14\u0026ndash;15]. They are known to inhabit primarily lowland wetland forests along rivers and coastal areas such as riverine, mangrove, peat swamp and freshwater swamp forests [13\u0026ndash;15]. They are folivores, feeding on a variety of plant parts including young leaves or shoots, unripe fruits, including seeds, and flowers [11, 14, 16, 17]. Having a multiple-chambered stomach, the proboscis monkey possesses unique digestive physiology that allows the anaerobic cellulolytic bacteria in their forestomach to ferment their food, and they are capable of regurgitating like the ruminants [16, 18]. Nevertheless, sugar-rich fruits are avoided as the fermentation process will increase acidity in their forestomach and cause bloating that could be fatal to them [14, 19, 20]. In addition, tree bark, termites and termite nest materials are also consumed, though infrequently, by the proboscis monkey for supplement mineral intake, used as a buffer to forestomach pH and removal of toxicity [14].\u003c/p\u003e \u003cp\u003eTo date, no information on gut microbiota is available for proboscis monkey populations from Sarawak. Previous research done by Hayakawa \u003cem\u003eet al.\u003c/em\u003e [21] focused solely on individuals from Sabah comparing various habitat types such as riverine, mangrove and man-made environment, whereas Jose \u003cem\u003eet al.\u003c/em\u003e [22] exclusively described the population from Menanggul River, Sabah. The foremost importance of the information on the gastrointestinal microbiome in proboscis monkeys across geographical regions is for a better interpretation of how different environments and habitat changes influence the diversity of the microbial community of that endangered and endemic species in Borneo. Moreover, the data is useful to assess the health status of the species as the gut microbiome plays an important role in the digestion processes and indirectly benefiting the immune system in many aspects. This can also facilitate the documentation of harmful bacteria present in the gastrointestinal tract of proboscis monkeys, which can be utilized to evaluate the potential of proboscis monkeys as a host reservoir for zoonotic diseases. Therefore, this study was designed to determine the diversity of gut microbiota in proboscis monkeys from Malaysian Borneo and the factors affecting the diversity (e.g., environmental factors and food source availability). It is hypothesised that populations living in natural habitats would possess higher diversity and abundance of gut microbial communities, enabling them to digest a wide range of dietary sources.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDiversity of proboscis monkeys\u0026rsquo; gut microbiota\u003c/h2\u003e \u003cp\u003eAmplicon sequencing resulted in a total of 2,741,163 paired-end sequencing reads of proboscis monkeys\u0026rsquo; 16S ribosomal RNA (rRNA) gut microbes with an average length of 436 base pairs. After the quality filtering processes, a total of 272,086 gut microbiota comprised of 4030 OTUs were discovered from the 24 faecal samples (24 individuals from 7 different localities). Overall, there are 98% of bacteria and 2% of archaea were classified in the analyses. The gastrointestinal bacteria comprised 340 species from 109 families and 20 phyla. Phylum Firmicutes dominated the bacterial phyla that cover 74% of total frequency, followed by Bacteroidetes (15%), and Verrucomicrobia, (3%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The distribution of the main bacterial phyla identified across seven different localities in Sabah and Sarawak is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. All localities showed a similar pattern with the highest percentage of Firmicutes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResults for all the seven sampling locations from Sarawak and Sabah are given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The number of observed OTUs is highest in Bako National Park, Sarawak (Shannon Index, \u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.015) and lowest in Klias, Sabah (Shannon Index, \u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.224). Despite the nature of the proboscis population from Lok Kawi which is kept in captivity, they sustain high gut microbial diversity (Shannon Index, \u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.127).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe summary statistics of proboscis gut bacteria from seven populations across Sabah and Sarawak.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBNP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKLS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKNM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLKW\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLMNP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSWS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of samples analysed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of families\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e175\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e111\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e107\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo. of individuals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38283\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32574\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e21162\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e36035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e42518\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShannon (\u003cem\u003eH\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.865\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.766\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.453\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSimpson\u0026rsquo;s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.9676\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.9136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9614\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.9602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9697\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.9469\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.9278\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003cb\u003e*\u003c/b\u003eLKW: Lok Kawi Wildlife Park; KLS: Padas Damit Forest Reserve, Garama, Klias; LB: Labuk Bay Proboscis Monkey Sanctuary; LMNP: Limbang Wetland National Park; KNM: Kuala Niah Miri; SWS: Samunsam Wildlife Sanctuary; BNP: Bako National Park.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the shift in the gut bacterial diversity across different populations (i.e., wild, semi-wild, and captive; refer to Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e), changes in Beta-diversity were compared, using the Weighted and Unweighted Unifrac distances (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The results show the segregation of gut microbial communities in different types of environments (Weighted: F\u0026thinsp;=\u0026thinsp;1.3286, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.177; Unweighted: F\u0026thinsp;=\u0026thinsp;1.3939, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049). The heatmap plot demonstrates major differences in the occurrence of the most abundant genera of the gut microbiota found in proboscis monkeys (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). There was a vast difference in the microbe composition that largely separated Lok Kawi from other populations, which signifies the changes in microbial diversity from captive proboscis monkeys, supported by the Bray-Curtis dissimilarity clustering shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Whereas the statistically significant Kruskal-Wallis \u003cem\u003eH\u003c/em\u003e test (\u003cem\u003eH\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14.70166, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022) supports the differences in the gut microbial composition of the proboscis monkey from various localities.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe proboscis monkey populations were observed in seven selected localities within Sarawak and Sabah.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEnvironmental conditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAccessibility\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSarawak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBako National Park (BNP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e- Mangrove, peat swamp, mixed dipterocarp forest, heath, beach and cliff vegetations.\u003c/p\u003e \u003cp\u003e- Wild populations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- Tourist spot.\u003c/p\u003e \u003cp\u003e- Very near to a village.\u003c/p\u003e \u003cp\u003e- Direct observation is available.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamunsam Wildlife Sanctuary (SWS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e- Mangrove and riverine forest.\u003c/p\u003e \u003cp\u003e- Wild populations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- Not accessible to public.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKuala Niah, Miri (KNM)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e- Mangrove forest.\u003c/p\u003e \u003cp\u003e- Wild populations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- Near to villages.\u003c/p\u003e \u003cp\u003e- Observation only available by boat.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLimbang Wetland National Park (LMNP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e- Mangrove forest.\u003c/p\u003e \u003cp\u003e- Wild populations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- Near to a village.\u003c/p\u003e \u003cp\u003e- Observation only available by boat.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSabah\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLabuk Bay Proboscis Monkey Sanctuary (LB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e- Mangrove and mixed dipterocarp forest.\u003c/p\u003e \u003cp\u003e- Semi-wild populations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- Tourist spot.\u003c/p\u003e \u003cp\u003e- Direct observation is available.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePadas Damit Forest Reserve, Garama, Klias (KLS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e- Mangrove and riverine forest.\u003c/p\u003e \u003cp\u003e- Wild populations.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- Accessible to public.\u003c/p\u003e \u003cp\u003e- Observation only available by boat.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLok Kawi Wildlife Park (LKW)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e- Captive.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e- Accessible to public.\u003c/p\u003e \u003cp\u003e- Direct observation is available.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe common bacterial taxa that are shared in all types of habitats comprise the fundamental bacterial community involved in the breakdown processes of complex plant materials, e.g.: Ruminococcaceae, Lachnospiraceae, and Muribaculaceae (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e listed the main bacterial taxa found from the seven populations of proboscis monkeys across Malaysian Borneo, dominated by phylum Firmicutes and Bacteroidetes. Firmicutes were characterized by two main orders, namely, Clostridia and Bacilli. Meanwhile, Bacteroidetes showed no changes in all populations with order Bacteroidia becoming the most significant representative. On the other hand, archaea were only represented by two families which are Methanobacteriaceae and Methanomethylophilaceae, the strictly anaerobes microorganisms. They are the significant methane producers through the metabolism of hydrogen and carbon dioxide.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison between the main bacterial phyla found in proboscis monkeys from seven localities.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLocality\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eMain gut bacterial phyla (and class) recorded\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBako National Park\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirmicutes\u003c/p\u003e \u003cp\u003e(Clostridia, Erysipelotrichia, Negativicutes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003cp\u003e(Bacteroidia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVerrucomicrobia\u003c/p\u003e \u003cp\u003e(Verrucomicrobiae)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProteobacteria\u003c/p\u003e \u003cp\u003e(Alphaproteobacteria, Gammaproteobacteria)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSamunsam Wildlife Sanctuary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirmicutes\u003c/p\u003e \u003cp\u003e(Clostridia, Erysipelotrichia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003cp\u003e(Bacteroidia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVerrucomicrobia\u003c/p\u003e \u003cp\u003e(Verrucomicrobiae)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProteobacteria\u003c/p\u003e \u003cp\u003e(Alphaproteobacteria)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKuala Niah, Miri\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirmicutes\u003c/p\u003e \u003cp\u003e(Bacilli, Clostridia, Negativicutes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003cp\u003e(Bacteroidia, Ignavibacteria)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVerrucomicrobia\u003c/p\u003e \u003cp\u003e(Verrucomicrobiae)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eActinobacteria\u003c/p\u003e \u003cp\u003e(Coriobacteriia)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLimbang Wetland National Park\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirmicutes\u003c/p\u003e \u003cp\u003e(Bacilli, Clostridia, Erysipelotrichia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003cp\u003e(Bacteroidia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVerrucomicrobia\u003c/p\u003e \u003cp\u003e(Verrucomicrobiae)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProteobacteria\u003c/p\u003e \u003cp\u003e(Alphaproteobacteria, Gammaproteobacteria)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLabuk Bay Proboscis Monkey Sanctuary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirmicutes\u003c/p\u003e \u003cp\u003e(Clostridia, Negativicutes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003cp\u003e(Bacteroidia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProteobacteria\u003c/p\u003e \u003cp\u003e(Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVerrucomicrobia\u003c/p\u003e \u003cp\u003e(Verrucomicrobiae)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePadas Damit Forest Reserve, Garama Klias\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirmicutes\u003c/p\u003e \u003cp\u003e(Clostridia, Erysipelotrichia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003cp\u003e(Bacteroidia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVerrucomicrobia\u003c/p\u003e \u003cp\u003e(Verrucomicrobiae)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCyanobacteria\u003c/p\u003e \u003cp\u003e(Melainabacteria)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLok Kawi Wildlife Park\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFirmicutes\u003c/p\u003e \u003cp\u003e(Bacilli, Clostridia, Erysipelotrichia, Negativicutes)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBacteroidetes\u003c/p\u003e \u003cp\u003e(Bacteroidia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProteobacteria\u003c/p\u003e \u003cp\u003e(Alphaproteobacteria, Deltaproteobacteria, Gammaproteobacteria)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVerrucomicrobia\u003c/p\u003e \u003cp\u003e(Verrucomicrobiae)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe findings of this study showed that Firmicutes bacteria constitute the highest proportion (73.5%) of the bacterial population, which aligns with previous research on Colobine monkeys which have consistently identified Firmicutes as the predominant phylum in the gut microbiota of these primates [23\u0026ndash;27]. The findings are also consistent with the research done on proboscis gut bacterial diversity in Sabah which found Firmicutes to be the most common phylum accounting for 16 to 82 percent of the bacterial composition [21\u0026ndash;22]. Firmicutes is a group of anaerobic bacteria, primarily characterised by their gram-positive nature. Notably, several species within this group possess the ability to create endospores, enabling their survival in harsh and extreme environments.\u003c/p\u003e \u003cp\u003eInterestingly, the lowest Firmicutes percentage recorded by Hayakawa \u003cem\u003eet al.\u003c/em\u003e [21] is only 16% of the individuals sampled in the semi-wild population (mangrove habitat, provisioned with artificial diet items). Meanwhile, this study documented consistent Firmicutes composition in all samples (including semi-wild population) which covers more than 60% of bacteria from every population. Even though the diet items supplied (vegetables cultivated for human consumption) for the semi-wild population comprised of high nutrient content, it drives the shift in bacterial diversity to human-like species composition [26, 28]. However, the comparison and interpretation of data must be done with caution as the study by Hayakawa \u003cem\u003eet al.\u003c/em\u003e [21] had only one sample representative from the semi-wild population, while this study analysed a total of four samples from similar habitat types.\u003c/p\u003e \u003cp\u003eThe Firmicutes bacteria in this study were represented by four major classes: Bacilli, Clostridia, Erysipelotrichia and Negativicutes. Nevertheless, class Clostridia dominated the Firmicutes composition covering 72% of the total frequency with family Ruminococcaceae and Lachnospiraceae as the most recorded families from that phylum. These two bacterial families are the bacteria that commonly occur in the mammalian gut microbiota, previously reported to have a high abundance in herbivorous animals and proven to have efficiency in the digestion of complex plant materials [29\u0026ndash;31]. This study clarifies the abundance of Ruminococcaceae and Lachnospiraceae in proboscis monkeys, which is consistent with the findings of Jose \u003cem\u003eet al.\u003c/em\u003e [22].\u003c/p\u003e \u003cp\u003eRuminococcaceae and Lachnospiraceae both assist in the degradation of complex polysaccharides into short-chain fatty acids and are capable of breaking down plant-derived substrate which includes cellulose, hemicellulose, and starch [30]. Species from the family Lachnospiraceae (\u003cem\u003eRoseburia sp., and Coprococcus sp.\u003c/em\u003e) produced high levels of butyrate significant as the energy source for the colon epithelium [32, 33]. On the other hand, the \u003cem\u003eEubacterium coprostanoligenes\u003c/em\u003e species (family Ruminococcaceae) that is found in this study carry an effective function as the cholesterol-reducing anaerobe, by converting cholesterol to coprostanol. The intestine is not able to absorb coprostanol and it will be excreted by the gastrointestinal system [34\u0026ndash;36]. Thus, it is proven that the record of bacteria from these two bacterial families would be a good indicator in assessing the health status of proboscis monkey populations.\u003c/p\u003e \u003cp\u003eNot only Firmicutes, Bacteroidetes also provide similar purposes in maintaining the health of proboscis monkeys and become part of important gut microflora in animals. This study recorded the family Muribaculaceae as the main representative from phylum Bacteroidetes, which are also commonly found in the guts of homeothermic animals, e.g., birds and mammals [37]. Covering the highest bacteria percentage after Firmicutes, Bacteroidetes is much needed by the host as it is specialized in degrading high molecular weight organic matter [38, 39].\u003c/p\u003e \u003cp\u003eThe primary driver of Muribaculaceae occurrence in the proboscis gut is due to its capability to degrade oxalate, complex carbohydrates (plant cell wall glycans), and encrypt elements of an electron transport chain for energy production. Oxalate and glycan are the main components of the plant cell wall that serve as the protection mechanism and support the energy metabolism of plants [40]. The proboscis monkey also benefited the plant glycan as a fundamental component of the protein folding process (glycosylation) for adaptive immune activation [41, 42]. Interestingly, the number of Muribaculaceae bacteria greatly decreased in the Lok Kawi population indicating captive population may have low varieties of food plant intake. The dietary sources of the proboscis monkey population in Lok Kawi solely depend on the food provided in the park. Thus, this finding is evidence of the fact that reduced natural food sources availability may account for the low abundance of gut bacteria observed in the captive population. Frankel \u003cem\u003eet al.\u003c/em\u003e [43] also documented a comparable finding in which folivorous NHPs housed in captivity, display alterations in their gut microbiomes when compared to the wild population. These changes have been attributed to the differences in dietary intake amongst NHP populations.\u003c/p\u003e \u003cp\u003eBesides that, similar to the findings of this study, Hayakawa \u003cem\u003eet al.\u003c/em\u003e [21] in their study of the semi-wild population reported the largest proportion of the genus \u003cem\u003ePrevotella\u003c/em\u003e, which belongs to the phylum Bacteroidetes. In addition to the natural food resources present in their habitat, the proboscis monkeys were provided with supplementary food sources, including vegetables (i.e., carrots and long beans). Consequently, this has resulted in a significant prevalence of \u003cem\u003ePrevotella\u003c/em\u003e species, which have been previously shown as the dominant members of the gut microbiota in both humans and captive primates [26, 28] The increase in \u003cem\u003ePrevotella\u003c/em\u003e abundance signifies a decline in the monkeys' capacity to metabolise dietary fibre, as \u003cem\u003ePrevotella\u003c/em\u003e primarily metabolises simple carbohydrates [27].\u003c/p\u003e \u003cp\u003eOther significant bacterial phyla detected in proboscis\u0026rsquo; faecal samples are the Verrucomicrobia, Proteobacteria, Cyanobacteria and Actinobacteria with each phylum covering at least 1% of bacteria of the overall microbial diversity. Verrucomicrobia and Proteobacteria are well portrayed in this study even though they are rarely described as microbiome components of non-human primate gut [25]. The occurrence of Verrucomicrobia, Cyanobacteria, and Actinobacteria in the gastrointestinal tract of the proboscis monkey is commonly attributed to the animal's intake of water. Those bacterial communities are highly prevalent in various environmental settings, frequently observed in both soil and water sources [44, 45]. In addition to inhabiting habitats characterised by aquatic environments, the proboscis monkeys were observed to partake of water sources in close proximity to their feeding grounds. This observation potentially elucidates the presence of bacterial communities within the gastrointestinal tract of the proboscis monkeys. Meanwhile, the detection of Proteobacteria was primarily characterised by the prevalence of species commonly linked to diseases in animals and humans. Hence, it demonstrates the capacity of the proboscis monkey to serve as a reservoir for pathogenic microbes, which will be further discussed below.\u003c/p\u003e \u003cp\u003ePathogen infection typically appears as asymptomatic, although it can lead to severe illness upon accumulation within the host organism. Previous research conducted on non-human primates has identified several pathogenic bacterial genera present in their gastrointestinal tract. These include \u003cem\u003eEscherichia, Salmonella, Mycobacterium, Campylobacter, Helicobacter, Pseudomonas, Shigella\u003c/em\u003e, and \u003cem\u003eYersinia\u003c/em\u003e [46\u0026ndash;50]. Likewise, the proboscis monkey is not exempt from this scenario and may potentially serve as a substantial reservoir for pathogenic microorganisms. This study identified various pathogens in the faecal samples obtained from the proboscis monkeys in different locations across Malaysian Borneo. Among the most significant findings are from the genera \u003cem\u003eCampylobacter\u003c/em\u003e, \u003cem\u003eHelicobacter\u003c/em\u003e, and \u003cem\u003eComamonas\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCampylobacter\u003c/em\u003e and \u003cem\u003eHelicobacter\u003c/em\u003e are known to be pathogenic to both humans and animals and suppress the host immune system by toxin secretion [51]. Previously, McKenna \u003cem\u003eet al.\u003c/em\u003e [48] recorded a high abundance of \u003cem\u003eCampylobacter\u003c/em\u003e bacteria in symptomatic animals compared to healthy individuals. Due to the sensitivity of the bacteria to the stomach's normal production of hydrochloric acid, low levels of exposure may not result in illness. However, the number of bacteria can swiftly increase as a result. Both Campylobacter and Helicobacter are likely prevalent in the proboscis monkey population, as they are abundantly present in all populations across Malaysian Borneo.\u003c/p\u003e \u003cp\u003eOn the other hand, the presence of \u003cem\u003eComamonas\u003c/em\u003e bacteria solely in the Lok Kawi region adds to the greater abundance of bacteria that are not shared with the semi-wild population. \u003cem\u003eComamonas\u003c/em\u003e is a prevalent pathogenic bacterium that is frequently encountered in many environmental settings and is known to be a causative agent of diseases in humans. The results obtained in the present study are consistent with previous research conducted by Amato \u003cem\u003eet al.\u003c/em\u003e [52] on Colobine monkeys where the captive population exhibited a higher abundance of pathogenic bacteria which heightened susceptibility to gut microbial dysbiosis.\u003c/p\u003e \u003cp\u003eIn addition to bacteria, the occurrence of archaea in the gastrointestinal tracts of humans, apes and ruminant animals has been documented in previous studies [28, 53, 54, 55]. However, there is a lack of comprehensive research on their metabolic roles. The methanogens are often encountered, and a comparable occurrence has been documented in proboscis monkeys, where 2% of the archaea population belongs to the families Methanobacteriaceae and Methanomethylophylaceae. The methanogens were able to endure in an environment devoid of oxygen and generate methane as a by-product through the process of reducing carbon dioxide using hydrogen or methanol. The archaea belonging to the Methanobacteriaceae family are frequently observed in the human gut and are commonly expelled from the body as flatus or by exhalation [54].\u003c/p\u003e \u003cp\u003eWith a compartmentalised stomach, the digestive physiology of the proboscis monkey resembles that of ruminants, and it utilises archaea as part of an important microbial community in food fermentation. Hydrogen, as a fermentation intermediate, will not accumulate in the digestive organ and will instead be metabolised by methanogenic archaea, releasing methane as a by-product [56]. Even though archaea comprise a small portion of the proboscis gut microbiota, they play an important role in the complex metabolic pathways of dietary fermentation in the GI tract of the proboscis monkey.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eStudies on the gut microbiota of non-human primates have proved the correlation of bacterial diversity and richness with various aspects that influenced the existence of microbes. Food source availability and diet changes had become the utmost important factors that shaped the microbiota diversity and abundance in the gut, besides the environmental components as a source of bacterial diversity [57]. The unique gastrointestinal physiology of Colobine monkeys allows them to consume fibre-rich foods that are hard to digest and are mostly unpalatable to other animals [58]. This survival strategy is made possible by the existence of diverse gut microbiota presence in their GI tract, and hence, becomes a critical aspect in assessing the health status of the monkey.\u003c/p\u003e \u003cp\u003eThe discussion of this study brings us to an understanding of the effects of habitat changes on gut microbial composition by evaluating data across different geographic locations and environmental conditions. Remarkably, as the first research done on proboscis\u0026rsquo; gut microbiome in Sarawak, information from this research provides an overview of the comparison of gut microbe species from across Borneo in comparison with proboscis data reported previously in Sabah [21\u0026ndash;22]. The shifts in the bacterial community were well described as being varied in different types of populations (i.e., wild, semi-wild, and captive), and the gut microbiome of the captive population of proboscis monkeys differed from other populations. In addition, the development of the Pan-Borneo Highway Project that traverses protected areas leads to deforestation and poses a significant risk to the proboscis monkey [59]. With the continuous issues of forest degradation and habitat loss, this Colobine monkey with a very specific diet preference might face problems with insufficient dietary supply. Thus, all the information and knowledge gathered from this study would be very beneficial for long-term planning on the conservation management of proboscis monkeys in Borneo.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eField sampling, sample collections and preservation\u003c/h2\u003e \u003cp\u003eField samplings were carried out at seven study areas within Sarawak and Sabah (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). \u003cb\u003eSarawak;\u003c/b\u003e Bako National Park (N 01\u0026deg;43.006\u0026rsquo;, E 110\u0026deg;26.650\u0026rsquo;), Samunsam Wildlife Sanctuary (N 01\u0026deg;57.159\u0026rsquo;, E 109\u0026deg;36.432\u0026rsquo;), Kuala Niah (N 03\u0026deg;55.855\u0026rsquo;, E 113\u0026deg;41.794\u0026rsquo;) and Limbang Wetland National Park (N 04\u0026deg;50.777\u0026rsquo;, E 115\u0026deg;00.104\u0026rsquo;). \u003cb\u003eSabah;\u003c/b\u003e Labuk Bay Proboscis Monkey Sanctuary (N 05\u0026deg;56.111\u0026rsquo;, E 117\u0026deg;48.242\u0026rsquo;), Lok Kawi Wildlife Park (N 05\u0026deg;50.584\u0026rsquo;, E 116\u0026deg;49.019\u0026rsquo;) and Padas Damit Forest Reserve, Klias (N 05\u0026deg;24.404\u0026rsquo;, E 115\u0026deg;33.180\u0026rsquo;). The data types of proboscis monkey\u0026rsquo;s habitat (i.e., wild, semi-wild, captive), and details on accessibility to every location were recorded for reference purposes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Non-invasive sampling technique was used by collecting the faecal samples to avoid any risk of harm to the species. Surveys on primate (to scout for the faeces) were done three times per day; morning (0630\u0026ndash;0900), afternoon (1100\u0026ndash;1300) and evening (1600\u0026ndash;1830). The faecal samples were collected immediately after defecation and stored in Qiagen RNAlater\u0026reg; Stabilization Solution.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA extraction, amplification, and amplicon sequencing\u003c/h3\u003e\n\u003cp\u003eMicrobial DNA were extracted using the QIAamp\u0026reg; Fast DNA Stool Mini Kit modified from the established protocol by QIAGEN Company. Extracted DNA products were sent to a private laboratory (Apical Scientific Sdn. Bhd.) for quality measurement. Amplification (PCR) of the V3-V4 region of the bacterial 16S rRNA gene was done using pyrotagged primers. The purity (ng/ul) and concentration (ratio absorbance at 260/280) of the amplicons were checked using Picogreen and Nanodrop. Samples were proceeded straight for library preparation using Illumina 16S Metagenomics Library Prep Kit and their quality and quantity were determined using Agilent TapeStation 4200. All libraries passed the QC measurement (DNA concentration and molarity of qPCR) and were pooled according to protocol by Illumina before sequencing was done using the MiSeq platform at 2x301PE format.\u003c/p\u003e \u003cp\u003e \u003cb\u003eData analyses\u003c/b\u003e \u003c/p\u003e\n\u003ch3\u003ea. Sequence Quality Filtering, Alignments and OTU Clustering\u003c/h3\u003e\n\u003cp\u003eData processing was done in QIIME (Quantitative Insight Into Microbial Ecology) software v2.1 [60]. Raw sequence reads were quality filtered, demultiplexed and denoised using DADA2 pipeline v1.6 [61], truncating reads shorter than 240 base pairs. Multiple sequence alignment was done using the MAFFT (Multiple Alignment using Fast Fourier Transform) program and a phylogenetic tree was inferred in FastTree v2.1.10. Sequences were then assigned to its Operational Taxonomic Units (OTUs) with a 95% similarity threshold against the bacterial database available in SILVA [62].\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eb. Microbial Species Diversity (Alpha and Beta Diversity)\u003c/h2\u003e \u003cp\u003eTo analyze the diversity of microbes between different groups, differential abundance testing was carried out by ANCOM (Analysis of Composition of Microbiome) method [63]. The alpha-diversity (within sample richness) and beta-diversity (between sample dissimilarity) were computed in R package v3.4.1 implemented in QIIME software, as well as PAST (Paleontological Statistics) software v3.0. For the alpha diversity, Shannon (\u003cem\u003eH\u003c/em\u003e) and Simpson\u0026rsquo;s Diversity Indices were used, while species evenness was calculated using Buzas-Gibson\u0026rsquo;s (E) Index. To reveal the factors influencing the diversity of the microbial community, Weighted UniFrac (quantitative) and Unweighted UniFrac (qualitative) distances were tested. The within-group and between-group variance were calculated using Permutational Multivariate Analysis of Variance (PERMANOVA) statistics in an R package with 999 permutations. The Bray-Curtis dissimilarity method and Kruskal-Wallis H test were also applied to observe the changes in gut bacterial diversity between proboscis monkey populations from different types of habitats.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eADDITIONAL INFORMATION\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e \u003cp\u003eThis research has been funded by the Ministry of Higher Education (MoHE) Malaysia under grants NRGS/1087/2013(01) and GL/07/UMS/03/2017 awarded to Associate Prof. Dr Faisal Ali Anwarali Khan and Dr Jaya Seelan Sathiya Seelan. Professor Dr Henry Bernard and research assistant was funded by research grant no. GKP0012-STWN-206 for fieldwork and data collection in Padas Damit Forest Reserve in Klias, Sabah.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eNM and FAAK contributed as the main researchers. HB, WNSWA, SS and MKZ support the team with fieldworks and sample collections. NM, FAAK, JSS, YLC and HB prepared the manuscript. All authors have read and agreed to the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003ePermit to conduct sampling activities were approved by Sarawak Forest Department (SFD) (Permit No. NCCD.907.4.4(JLD.13)-265) and Sabah Biodiversity Centre (SaBC) (Permit No. JKM/MBS.1000-2/2 JLD.7 (130)). Thank you to Molecular Genetics lab members of Universiti Malaysia Sabah for providing equipment to conduct lab works in Sabah. Deepest appreciation to all Sarawak Forestry Corporation (SFC) staffs and UNIMAS Primates Research Team for their endless helps during the sampling trip. Finally, we would like to express gratitude to the researchers at Queen Mary University of London (QMUL) for their guidance in conducting the bioinformatics analysis.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in the National Genomics Data Centre, China National Center for Bioinformation (GSA Accession No: CRA016781) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePoirier, F. E., \u0026amp; Hussey, L. K. Non-human primate learning: The importance of learning from an evolutionary perspective. \u003cem\u003eAm Anthropol Assoc\u003c/em\u003e, \u003cstrong\u003e13(2)\u003c/strong\u003e, 133\u0026ndash;148 (1982).\u003c/li\u003e\n\u003cli\u003eSuomi, S. J. 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Analysis of composition of microbiomes: A novel method for studying microbial composition. \u003cem\u003eMicrob Ecol Health Dis\u003c/em\u003e, \u003cstrong\u003e26(1)\u003c/strong\u003e, 27663 (2015).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Borneo, metagenomics, microbiome, primates","lastPublishedDoi":"10.21203/rs.3.rs-4487731/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4487731/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNumerous research on the gut microbial diversity of non-human primates (NHP) has been done to evaluate the impact of environmental changes on the diversity of gut microbiota and to determine the mutualistic relationship between the animals and gut microbes that aid in their adaptation to living in an altered environment. The metagenomics approach was employed to explore gut microbial diversity and abundance in the endangered and endemic proboscis monkeys of Borneo. The amplicon sequencing of the 16S ribosomal RNA (rRNA) gene from 24 faecal samples successfully generated over two million raw reads of bacteria (98%) and archaea (2%) representing 4030 OTUs. Gut microbial diversity and abundance vary across different populations, i.e.: wild, semi-wild and captivity, suggesting that higher diversity was discovered in individuals inhabiting areas with higher food resource availability in natural habitats. The observed alterations in gut microbial diversity and abundance among proboscis monkey populations inhabiting distinct environmental conditions in this study provide empirical evidence for the impact of environmental changes on the proboscis monkey\u0026rsquo;s gut microbiota. Besides, the prevalence of pathogenic bacteria identified in the proboscis monkey\u0026rsquo;s gut highlights the potential hazards associated with human-primate interactions and raises the urgent need for public health management. \u003cb\u003e[197 words]\u003c/b\u003e\u003c/p\u003e","manuscriptTitle":"Unravelling the metagenomic landscape of gut microbiota in endangered proboscis monkey across Malaysian Borneo: Conservation and ecological insight","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-26 19:00:30","doi":"10.21203/rs.3.rs-4487731/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ce80eee-6b61-49e5-b3f9-e2a2fc98ce7f","owner":[],"postedDate":"June 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33182253,"name":"Biological sciences/Genetics/Microbial genetics"},{"id":33182254,"name":"Biological sciences/Ecology"}],"tags":[],"updatedAt":"2024-08-13T03:06:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-26 19:00:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4487731","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4487731","identity":"rs-4487731","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}