Analysis of winter diet in Guizhou golden monkey (Rhinopithecus brelichi) using DNA metabarcoding data

The Guizhou golden monkey (Rhinopithecus brelichi), also known as the grey snub-nosed monkey, is critically endangered and found exclusively in China's Fanjingshan National Nature Reserve (FNNR). Understanding its dietary composition is crucial for assessing its feeding strategies and informing conservation plans. Previous studies relied on methods such as observation, stomach content analysis, and fecal microscopy, which are limited in their ability to identify dietary components with precision, especially in elusive species like the Guizhou golden monkey. The study's primary goal was to use the advanced DNA metabarcoding technique to address three key questions: (1) identifying the main dietary components of R. brelichi during winter, (2) determining the plant habits consumed, and (3) discovering any previously unreported food items. This approach offers a significant advantage over traditional methods by providing detailed taxonomic information with high sensitivity and throughput, allowing researchers to comprehensively characterize even cryptic elements in the monkey's diet. This research hypothesized that: (1) R. brelichi predominantly consumes shrubs and herbs in winter; (2) plant diversity in their diet surpasses animal diversity; and (3) previously undocumented food sources exist for this species.

Prior research on R. brelichi's diet has mainly utilized observational studies and fecal microscopy. These studies have indicated a seasonal variation in diet, with leaves, buds, flowers, and fruits being major components throughout the year. Winter diets, in particular, have been reported to consist largely of tree buds, bark, and flower buds, suggesting that food scarcity may be a factor during this period. However, these methods have limitations in accurately identifying dietary components at finer taxonomic levels. The use of DNA metabarcoding represents a significant advancement in dietary analysis, offering increased resolution and the potential to identify even small, cryptic components of the diet. Several studies have already employed this technique successfully in other primate species, providing insights into plant and animal consumption. The current research aims to leverage this technology to obtain a more comprehensive and detailed understanding of the R. brelichi's winter diet.

This study obtained ethical approval from the FNNR Administration. Fecal samples (n=31) were collected from December 2021 to January 2022 within the FNNR, with care taken to avoid disturbing the monkeys. Sample collection sites were recorded, noting GPS coordinates and vegetation type. Samples were preserved in ethanol and stored at -80°C. DNA extraction was performed using an OMEGA kit. The chloroplast rbcL and mitochondrial COI genes were amplified using specific primers and sequenced via Illumina NovaSeq PE250 platform. Bioinformatic analysis involved quality control (FastQC, Trimmomatic), sequence merging (FLASH), chimera removal (QIIME2), and OTU clustering at 97% similarity. Taxonomic assignment was conducted using BLASTN against the NCBI database, with manual verification using available FNNR flora and fauna data. Dietary composition was calculated using the percentage of occurrence (%RO) method. Plant genera were categorized into habits (herbs, shrubs, trees, lianas), and diversity indices (Shannon, Simpson) were calculated using the iNEXT package to assess dietary diversity. Sunburst charts and proportional diagrams were generated using Origin 2022 to visualize dietary composition.

DNA metabarcoding of 31 fecal samples revealed a diverse winter diet for R. brelichi. At the family level, the most abundant plant families were Magnoliaceae, Rubiaceae, Lauraceae, Adoxaceae, and Rutaceae, while at the genus level, Magnolia, Morinda, Viburnum, Tetradium, and Eurya were most prevalent. Plant food habit analysis showed shrubs/trees (45% abundance, 55% occurrence) dominated, followed by herbs and lianas. Analysis of animal food revealed Psychodidae, Trichinellidae, Staphylinidae, Scarabaeidae, and Trichoceridae as the most abundant families. Dietary diversity analysis showed significantly higher species richness and diversity in plant food compared to animal food. Several plant and animal species identified have not been previously reported in the R. brelichi diet, including Holboellia latifolia, Persicaria maculosa, Cinnamomum camphora, Glyptostrobus pensilis, and Menyanthes trifoliata.

The findings support the hypothesis that R. brelichi primarily consumes shrubs and herbs during winter, likely utilizing fallback foods due to decreased availability of preferred resources. The higher diversity in plant food sources compared to animal food aligns with the observed dominance of folivorous habits in this species. The identification of several novel food items highlights the power of DNA metabarcoding in uncovering previously unknown aspects of the Guizhou golden monkey's diet. The reliance on shrubs and fallback foods during winter necessitates conservation strategies focused on maintaining the abundance and diversity of these plant communities within the FNNR. The discovery of new food items also underscores the need for more extensive surveys to fully characterize the dietary landscape available to this endangered species. The limitations of species-level identification using DNA metabarcoding are acknowledged, with the potential for pseudogenes and heterozygotes impacting accuracy. Future research should explore the use of more comprehensive methods, such as metagenomics, and create local reference databases to enhance species identification accuracy.

This study provides the first comprehensive assessment of the winter diet of R. brelichi using DNA metabarcoding, revealing a diet dominated by Magnoliaceae, Rubiaceae, Adoxaceae, and diverse insects. The findings highlight the importance of both plant and insect resources in the winter diet and underscore the need for conservation strategies that protect diverse plant communities. Future studies should utilize improved methods for species-level identification and explore seasonal dietary variations to better inform conservation efforts.

The study's main limitation is the reliance on fecal DNA metabarcoding for dietary analysis. While this method offers enhanced resolution compared to traditional techniques, the accuracy of species-level identification can be affected by factors such as the presence of pseudogenes and heterozygotes in the amplified DNA. The incomplete nature of reference databases also limits the identification of certain species. Furthermore, the study focuses only on winter diet; broader seasonal studies are needed to fully understand the dietary flexibility of the Guizhou golden monkey.