The takin (Budorcas taxicolor) is a large hoofed bovid herbivore of the Himalayan region, belonging to the Bovidae family and Caprinae subfamily (Li et al., 2022). The species comprises four subspecies, namely the golden takin (Budorcas taxicolor bedfordi), Sichuan takin (Budorcas taxicolor tibetana), Bhutan takin (Budorcas taxicolor whitei), and Mishmi takin (Budorcas taxicolor taxicolor), distinguished according to their morphology and geographical distribution (Chen et al., 2017). The Sichuan takin and Golden takin are unique to China (Zhu et al., 2018); meanwhile, B. taxicolor has been listed as an endangered and vulnerable species by the International Union for Conservation of Nature (IUCN) (Ju et al., 2021). Like many wild animals, wild B. taxicolor is under pressure, particularly due to hunting and habitat loss caused by rapid population growth (Santymire et al., 2021).

The digestive tract of animals harbors myriad microorganisms (Xue et al., 2015). Indeed, the gastrointestinal tract (GIT) is one of the most complex and rapidly evolving biotic networks responsible for animal functional health (Al Jassim and Andrews, 2009). Fungi contribute to dietary changes by producing cellulolytic and hemicellulolytic enzymes that enhance the capacity for digesting leaves. However, the feces microbiome also affects immune responses by inhibiting or promoting local inflammatory responses (Iliev et al., 2012). Although intestinal fungi are closely associated with animal health, the composition and diversity of intestinal fungi remain unknown for many herbivores.

According to anatomical records from the Chengdu Zoo, the B. taxicolor is similar in size to the wild yak (Bos grunniens) and gaur (Bos gaurus), while its digestive system has characteristics similar to those of sheep. The Sichuan takin is a social ruminant, feeding primarily on trees and shrubs (Kang et al., 2018). As a large ruminant, B. taxicolor has a unique digestive system comprising four gastric cavities and relatively long lower feces, suitable for feed rich in plant cell walls (Zhu et al., 2018). The bulk of current literature addresses only ruminant fermentation (Mura et al., 2019). Although the bacterial community composition of B. taxicolor has received considerable research attention, the characterization of fungal colonization has been relatively neglected (Guan et al., 2023; Ma et al., 2023).

Although the microbiome composition and functions remain stable over extended periods, a multitude of factors significantly impact these properties. These include but are not limited to dietary factors, geographical location, age, genetics, mode of delivery, and medical treatments (Panda et al., 2014). Accordingly, we hypothesize that the composition of intestinal fungi may be related to geographical location or diet. To test this hypothesis, we analyzed the composition and potential pathogens in the intestinal feces of B. taxicolor. The findings of this study provide new insights into the composition of intestinal fungi while supplementing existing knowledge on their assumed role in health. The finding will help protect this species by providing vital information.

Budorcas taxicolor is a class I protected animal in China that is listed as an endangered species in the Red Book. To date, China has taken various protective measures, including establishing nature reserves and implementing captive breeding. However, these strategies have ignored the effect of intestinal fungi in wild Takin.

In the current study, data was analyzed based on the RDP database, which provides 28S rRNA sequences for Fungi (Eukarya) (Balvočiūtė and Huson, 2017). RDP has +97% assignment accuracy and provides rapid results for reads that are 250 bp or longer when they originate from a taxon known to the database (Lan et al., 2012). Indeed, the RDP classifier has been employed to correlate mammals to their gut microbes (Ley et al., 2008). However, in the current study, most annotations were limited to genera due to the short reads (<250 bp), impeding the investigation of detailed species. Moreover, fungi have received relatively less research attention. In our study, the fraction of non-zero values was only 0.033. Additionally, given that fungi account for ~0.1% of the gut (Qin et al., 2010; Arumugam et al., 2011), many fungal classifications have not yet been annotated in the RDP database. This limitation hinders research, particularly in non-human species. Accordingly, the amplicon sequencing of ITS rRNA results were verified by amplicon metagenomic sequencing and isolation cultures. Aspergillus and Trichosporon from isolation culture and amplicon metagenomic sequencing validated the ITS rRNA sequencing results.

Alpha diversity analysis was employed to evaluate and compare the species diversity of individual plots. As the curve becomes flat, the quantity of sequencing data tends to be reasonable, and the homogeneity among samples is high (Kleine Bardenhorst et al., 2022), indicating that the sample size is adequate for data analysis (Ma et al., 2021). The differences in the Simpson and Shannon indices between the five groups were significant (p < 0.05). GII had the highest abundance, indicating the highest species richness and diversity among the five groups. The AVS count of GII was the highest (Figure 1), which could be attributed to the location and diet. Similar dietary effects were also demonstrated by Zhu et al. (2018) in their analysis of fecal microbial diversity in captive and wild B. taxicolor antelope. Moreover, the fungal diversity within the feces of wild B. taxicolor was highest in Ascomycota (82.19%) at the phylum level. Similarly, Ascomycota contributed the highest proportion of core feces microbial communities in sheep (Tanca et al., 2017), giraffes (Schmidt et al., 2018), Cervus nippon (Hu et al., 2020), and Tibetan macaques (Sun et al., 2018). This phenomenon may arise from Ascomycetes being primarily plant parasites. Additionally, Ascomycota is influenced by fiber content (Pitta et al., 2010). The environment at different sites likely differ regarding food, water, and soil composition. Additionally, certain conservation stations have salt ponds, which may have contributed to the observed differences by increasing the soil salinity (Supplementary Figures S3, S4) (Li et al., 2016).

Twenty-five fungal genera were identified, some of which were potentially pathogenic. When animals age, parasitic infections, traumatic injuries, and other diseases cause their immune function to decrease, making them susceptible to infection by potentially pathogenic fungi. All five groups of samples contained Thelebolus, which was the genera; similar findings were reported in yak (Zhu et al., 2023) and beef cattle (Tong et al., 2023) under different feeding patterns. Thelebolus negatively impacts ruminant health and behavior (Koester et al., 2020). Mechanistically, Thelebolus spp. may influence exopolysaccharide to further induce apoptosis in healthy intestinal cells, thus restricting nutrient uptake and decreasing intestinal absorption.

In addition, Cryptococcus and Trichosporon were detected in our study samples. Cryptococcus (de Freitas et al., 2020) and Trichosporon (Centeno-Martinez et al., 2023) have also been detected in beef cattle. Cryptococcal meningitis may occur in animals infected with cryptococcus (Yu D. et al., 2021), whereas multiple Trichosporons, including Trichosporon asahii (Ramírez and Moncada, 2020), can cause fatal Trichospore bacteremia (Basu et al., 2015). Furthermore, Zopfiella and Podospora were also detected in our study samples. Zopfiella (Fujimoto et al., 2004) and Podospora (Ying et al., 2021) cause immune suppression. However, the isolation of potentially pathogenic fungi from feces does not provide definitive evidence of a host’s health status (Ma et al., 2022). Moreover, although the distribution of these pathogens in the wild B. taxicolor is unknown, they might represent a source of zoonotic infections. However, due to the relative dearth of relevant data, the roles of the fungal community and individual fungal taxa in intestinal physiology remain poorly understood (Hu et al., 2020). In the last 20 years, considerable progress has been made in the study of mammalian intestinal tissue; however, research on the function of microorganisms in the lower feces of ruminants remains at a relatively early stage (O’Hara et al., 2020).

When an animal’s immune system is compromised, opportunistic fungi can become pathogenic. In some cases, the risk of infection increases, especially in a stressed state during which external factors exert adverse effects on the body’s immune system. However, the microbial ecology of feces does not precisely reflect the health of the host as certain animal pathogens are not responsible for causing B. taxicolor disease. On the one hand, no targeted etiological studies have been carried out to identify and verify the pathogenesis of these potential pathogens. On the other hand, capturing this animal for further investigation and confirmation has proven impossible for our research team. Nevertheless, this point of view can be put forward for the scientific protection of B. taxicolor (Ma et al., 2022).

The comprehensive analysis of KEGG metabolic pathways indicated that most microorganisms were predominantly involved in processes related to replication repair, translation, and nucleotide metabolism. However, others were associated with carbohydrate metabolism, fat metabolism, and energy metabolism. These metabolic processes are an important factor in determining fungal pathogenicity and virulence, due to their essential roles in facilitating the onset and advancement of infectious processes and in governing the generation and functionality of virulence factors, such as the fungal cell wall structure and melanin (Ruijter and Visser, 1997; ter Schure et al., 2000; Ries et al., 2018). The metabolic processes in these fungi significantly contribute to their adaptability, survival, and virulence within their host environment (Ries et al., 2018).

In this study, we used amplicon sequencing of ITS rRNA to comprehensively analyze the fungal diversity in the feces of wild B. taxicolor from five conservation stations. The fungal diversity primarily comprised Ascomycot, Fungi_unclassified, and Basidiomycota at the phyla level. Most microorganisms were found to have critical roles in replication, repair, translation, and nucleotide metabolism. The GII site had the highest species richness and diversity among the five groups, which may be due to differences in location and diet. We also detected potentially pathogenic genera, including Thelebolus, Cryptococcus, Trichosporon, Zopfiella, and Podospora, providing forewarning to inform the conservation of these endangered species. Although the fecal samples were relatively large, capturing wild B. taxicolor was not possible, impeding the evaluation of clinical symptoms and, thus, preventing an in-depth analysis of host health. Future studies should focus on evaluating a larger sample of B. taxicolor in terms of study duration and area to achieve a more comprehensive comparative analysis. Nevertheless, our study is the first step toward elucidating the fungal diversity in the feces of wild B. taxicolor.

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 in the article/Supplementary material.

The animal study was approved by Sichuan Agricultural University Animal Ethics Committee and Sichuan Tangjiahe National Nature Reserve (permit number DYYS20211118). The study was conducted in accordance with the local legislation and institutional requirements.

XM: Formal analysis, Funding acquisition, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. ZoL: Data curation, Formal analysis, Software, Visualization, Writing – original draft. LC: Funding acquisition, Project administration, Resources, Validation, Writing – original draft. MX: Conceptualization, Formal analysis, Investigation, Resources, Writing – original draft. FH: Formal analysis, Investigation, Methodology, Resources, Writing – original draft. ZnL: Methodology, Validation, Visualization, Writing – original draft. DC: Conceptualization, Investigation, Software, Writing – original draft. YW: Investigation, Methodology, Supervision, Writing – review & editing. LS: Funding acquisition, Project administration, Resources, Writing – review & editing. YG: Conceptualization, Data curation, Formal analysis, Supervision, Validation, Writing – review & editing.