In the face of enormous ecological pressures resulting from human disturbance, nonhuman primates (hereafter NHPs) are experiencing a severe population decline and the threat of extinction on a global scale (Estrada, 2013; Estrada et al., 2017). As an important contributing factor, anthropogenic disturbance often leads to a decrease in the availability and quality of food resources and increased social stress, negatively impacting individuals’ fertility and health (Almeida-Rocha et al., 2017). Recent studies also indicate that anthropogenic disturbance can result in significant changes in host gut microbial community composition including the loss of microbial diversity (Junge et al., 2011; Amato et al., 2013; Barelli et al., 2020). Given that the gut microbiome plays a crucial role in host nutrition, immune systems, and health (Flint et al., 2012; Nicholson et al., 2012; Fung et al., 2017), understanding the effects of anthropogenic disturbance on host gut microbial composition is critical for developing effective conservation strategies for environmental restoration and protecting threatened and declining species (Stumpf et al., 2016; Cavada et al., 2019).

Nonhuman primates that are housed in captive environments (e.g., zoos, rehabilitation and breeding centers) experience changes or restrictions in diet, increased exposure to human-associated microbes, and increased social stress, and are subject to medical interventions including treatments with antibiotics (McKenzie et al., 2017). A number of studies have shown that these changes are associated with the dysbiosis of the host gut microecosystem, including a loss of bacterial diversity (Amato et al., 2013; Clayton et al., 2016; McKenzie et al., 2017; Frankel et al., 2019), a reduction of native gut microbial taxa (Clayton et al., 2016), and an increase in antibiotic resistance genes of the gut microbiome (Tsukayama et al., 2018). Recent studies have shown that providing captive animals with more natural diets or releasing captive animals back into their natural habitat can help restore their native gut bacteria (Schmidt et al., 2019; Liddicoat et al., 2020; Martínez-Mota et al., 2020).

Our understanding of the NHP gut microbiome is based principally on the bacterial component. Less is known about the function and composition of fungal communities in the gut (also known as the mycobiome), especially the effects of environmental change on mycobiome composition. Fungi play critical roles as decomposers, mutualists, and pathogens in a range of ecosystems (Tedersoo et al., 2014). In addition to being a potential pathogen, there is mounting evidence that fungi in the gut serve as an important function in altering gut bacterial composition (Huffnagle and Noverr, 2013; Getzke et al., 2019), modulating host innate and adaptive immune responses (Wüthrich et al., 2012; Rizzetto et al., 2015), and biomass-degrading of host diet (Solomon et al., 2016). In humans, dysbiosis of the gut mycobiome is closely associated with host metabolic and immune disorders, colorectal adenomas, and inflammatory bowel diseases (IBDs; Luan et al., 2015; Mar Rodríguez et al., 2015; Wheeler et al., 2016; Sokol et al., 2017). A recent study found that lab mice released into a natural environment experienced a change in gut mycobiome and an improvement in host immune function (Yeung et al., 2020). Moreover, Barelli et al. (2020) found that the mycobiome community composition differed considerably between primate species (Procolobus gordonorum and Papio cynocephalus) living in protected and fragmented habitats (Barelli et al., 2020). However, for most primate species, the effects of captivity on host gut mycobiome and its potential impact on host health, remain unknown. In this regard, studies of the gut mycrobiome of individuals living under a range of wild, semi-natural, and captive conditions offer valuable perspectives and tools for investigating and monitoring critical links between diet, environment, and health that can inform captive management, reintroduction programs, and conservation strategies (Stumpf et al., 2016; Trevelline et al., 2019; West et al., 2019).

In the present study, we examined the mycobiome of Tibetan macaques (Macaca thibetana), a Near Threatened primate species endemic to China. We compared individuals living in three different environmental settings: captivity (Tong Ling City Zoo), a free-ranging and semi-provisioned group that is visited by tourists (Mt. Huangshan), and a wild group (Mt. Tianhu). In comparing the mycobiome of individuals in each of these three settings, we addressed three main questions: (1) Are there significant differences in gut fungal diversity among captive, semi-provisioned, and wild populations of Tibetan macaques? Secondly (2) What do differences in the composition and ecological function of the gut mycobiome tell us about the role of fungi in the primate digestive tract? and (3) What are the potential factors that shape mycobiome composition in the gut? We present these results in the context of what is known about the mycobiome in other mammals and discuss the potential impacts of captivity on the primate gut mycobiome.

In the current study, we found that the alpha diversity of the mycobiome of individuals in a captive group of Tibetan macaques translocated from their natural habitat 1 year ago and was reduced compared to individuals in two wild-living groups. One of the wild groups was semi-provisioned. This result is consistent with previous studies on the gut bacterial microbiome of NHPs (Clayton et al., 2016; McKenzie et al., 2017; Frankel et al., 2019). Controlled studies of humans indicate that the loss of gut fungal diversity is associated with susceptibility to diseases, including IBDs (Sokol et al., 2017). In other mammals, a loss of gut fungal diversity has been shown to result in a decrease in the ability to digest cellulose, and a weakend immune response (Akin et al., 1983; Denman and Mcsweeney, 2006; Sokol et al., 2017; Yeung et al., 2020). Therefore, lower alpha diversity among the captive Tibetan macaques has important implications for primate welfare and may be an early indication of negative health consequences resulting from captivity. Future studies are needed to assess the degree to which loss of gut fungal diversity affects the health of captive primates.

Our results also indicated a marked difference in beta diversity between the semi-provisioned group and the completely wild groups, but no difference in alpha diversity, despite the fact that the HS group has several decade of ecotourism and approximately one-third of their diet is provisioned (Li and Kappeler, 2020). This result is consistent with previous finding in two tropical primate species, one living in a protected forest and the other living in a fragmented forest (Barelli et al., 2020). In addition, it has been reported that lab mice exposed to a natural environment to have a more diverse mycobiome and that should provide positive consequences for host’s immune function (Yeung et al., 2020). Our results together with recent finding revealed that living in the wild and consuming a diverse diet are beneficial to maintaining gut fungal diversity of NHPs, while living in captivity, consuming a less varied diet, and limited exposure to soils and wild plants are expected result in a significant reduction in gut fungal diversity.

Previous studies have implied that fiber-rich diets may stimulate gut fungal diversity. For example, in ruminants, individuals consuming a diet high in plant fiber were found to exhibit higher gut fungal diversity compared with individuals fed a diet lower in plant fiber, such grains (Denman and Mcsweeney, 2006; Denman et al., 2010; Boots et al., 2013). Although, we do not have quantitative data on the fiber content of the wild plant foods consumed by the wild and semi-provisioned macaques, they naturally consume a broad-based diet that included grasses, leaves, fruits, and insects (Huffman et al., 2020). However, the main diet of captive group was corn supplemented with sweet potatoes. Corn fed to the captive macaques contains approximately 9 g of dietary fiber per 100 g consumed, and fiber in sweet potatoes contains approximately 10 g of dietary fiber per 100 g consumed (Sun et al., 2014). A previous study revealed that the crude fiber content of the major components of the plant diet of wild living Tibetan macaques ranged from 17.91 to 47.92% (You et al., 2013). Lower dietary fiber in the foods consumed by the captive macaques offers a possible explanation for their decreased gut mycobiome diversity compared to the wild and semi-provisioned free-ranging macaque population.

In addition to diet, other environmental sources of gut fungi include the soil (Fiers et al., 2019; Richard and Sokol, 2019; Mann et al., 2020). Given the conditions of their captive environment, which included frequent cleaning of concrete surfaces and metal bars, access to soil fungi was extremely limited. In this regard, we found that the relative abundance of the two main fungal guilds of plant pathogens and ectomycorrhizal fungi was decreased significantly in captive individuals compared to individuals living in both wild groups. Plant pathogenic and ectomycorrhizal fungi are two dominant guilds naturally found in plant and soil (Averill et al., 2016; Yang et al., 2019). Although our data showed a marked difference in mycobiome composition between the semi-provisioned free-ranging group and the un-provisioned wild group, mycobiome diversity between these groups did not differ. This pattern suggests that the free-ranging group was not exposed to fewer fungi but rather than differences in the environment and diet resulted in exposure to distinct fungi and/or altered selective pressure on the existing fungal community in the macaque gut.

We also found that the gut mycobiome of macaques in all three study groups was dominated by two phyla Ascomycota and Basidiomycota. This is consistent with previous studies in humans and NHPs (Hallen-Adams et al., 2015; Strati et al., 2016; Suhr et al., 2016; Wheeler et al., 2016; Sokol et al., 2017; Sun et al., 2018). Our results demonstrated that several fungal taxa differed in relative abundance, at the levels of genus, family, order, class, and phylum, among our three study groups. As described for fungal diversity, this finding may be a result of diet since recent studies of the human gut mycobiota have found the abundance of gut fungal taxa fluctuates with host diet. For example, plant-based diets (rich in grains, legumes, fruits, and vegetables) were associated with enrichment in Candida, whereas individuals consuming animal-based diets (rich in meats, eggs, and cheeses) were enriched in Debaryomyces and Penicillium (David et al., 2014; Hallen-Adams and Suhr, 2017). In particular, the zgenus Trichosporon (relative abundance, 5.82%; occurrence rate 100%) was assigned to the guild of potential animal pathogens based on FUNGuild analyses. Several species in this genus are responsible for the majority of cases of trichosporonosis, which are important opportunistic pathogens of humans (Marty et al., 2003; Colombo et al., 2011). However, although captivity may increase disease risk in primates, it is unclear if captive Tibetan macaques in this study showed a compromised immune function.

Interestingly, the genera Penicillium and Aspergillus, which were overrepresented in the captive individuals, also were enriched in the semi-provisioned free-ranging individuals compared to the wild individuals. These two genera, commonly reported as core genera in human gut mycobiome studies, also are found in foods consumed by humans and prone to spoilage (Suhr and Hallen-Adams, 2015; Hallen-Adams and Suhr, 2017). Although varying in degree, corn was a major component of the diet of both the captive and semi-provisioned free-ranging populations. Thus, we speculate either that a corn-derived fungi or possible transmission of gut fungi between humans handling the corn and the monkeys, may have promoted the increase of these two fungal genera.

Tibetan macaques in the wild and semi-provisioned groups had access to fungi naturally occurring in the soil and in animals and plants consumed. We found that the two main fungal guilds of plant pathogen and ectomycorrhizal fungi, which are commonly reported in plant or soil mycobiome, were decreased significantly in captive individuals (Averill et al., 2016; Yang et al., 2019). In addition, Cladosporium which was overrepresented in the wild group, and Trichoderma and Talaromyces, which characterized the semi-provisioned free-ranging group, have been isolated from soil and organic matter such as decaying wood and fruit (Potin et al., 2004; Chen and Zhuang, 2017; Salomão, 2018). Therefore, the presence and availability of fungi associated with foods and soil in the natural environment offer a likely explanation for certain specific differences in the gut fungal composition of wild and free-ranging macaques compared to captive individuals (Fiers et al., 2019; Richard and Sokol, 2019; Mann et al., 2020). However, a lack of information about metabolic pathways characterizing gut fungi and the influence of diet and environment in mycobiome composition and function, impact our ability to fully interpret these results.

Our findings demonstrate that individuals translocated from their natural habitat and fed a highly monotonous diet in captivity for 1 year, were characterized by reduced diversity and increased potential fungal pathogens in their gut. Future studies should focus on investigating whether the predominant gut fungal taxa detected here are food-borne travelers or symbiotic residents under modulation by the host and diet. To that end, culture-dependent and metagenomic approaches, along with mapping interactions between gut fungi and gut function are warranted. Additionally, it is essential to fully characterize the dietary behaviors and environmental exposures of primate populations to infer seeding mycobiome sources and the potential impact of gut fungi on primate health. Such data can be used to devise management strategies that can modulate the mycobiome of wild NHPs living in degraded habitats, and the health and longevity of captive primates that may be reintroduced into the wild.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI, PRJNA701719.

BS and JL conceived and designed the experiments. BS, YX, XX, WL, MH, XW, and DX performed the experiments. BS, YX, XX, PG, KA, and AG contributed to reagents, materials, and analysis tools. BS, YX, XX, PG, KA, AG, and JL wrote the paper. All authors reviewed the manuscript and agreed to the published version of the manuscript.