The genus Presbytis is a diverse primate from the Old World Monkey (Meyer et al. 2011), with the largest number of species amongst the subfamily Colobinae (Family: Cercopithecidae) (Roos et al. 2014). There are five species of Presbytis recorded in Sarawak and Presbytis chrysomelas is a unique species endemic to Borneo (Md-Zain et al. 2022, Noor-Faezah et al. 2023, Nur-Aizatul et al. 2024). There are two subspecies of P. chrysomelas — P. c. cruciger and P. c. chrysomelas (Roos et al. 2014); both of these subspecies have a distinct colour morphology (Groves 2001, Ampeng et al. 2024); however, historical records of mixed-troops of both subspecies suggest that their variation should be considered as colour variants rather than different subspecies (Phillipps and Phillipps 2018). Unlike P. c. chrysomelas with two colour variations (black and white) (Ampeng 2003), P. c. cruciger has three colour variations: head, shoulders, sides of the abdomen, thigh and calves have red–orange hair; hair on its cheeks, under its chest and abdomen are white; and arms, hands, feet and lines on the back are black (Rifqi et al. 2019, Ampeng et al. 2024).

P. c. cruciger is endemic to Sarawak and Kalimantan, along with its sister taxon, P. c. chrysomelas and has been sighted in Maludam (Sarawak) and Danau Sentarum (Kalimantan) (Phillipps and Phillipps 2018, Rifqi et al. 2019, Santoso et al. 2023a, Santoso et al. 2023b). Although its distribution is confined to Maludam, no comprehensive scientific information is available. Recently, Ampeng et al. (2024) made an important discovery about the first sighting of P. c. cruciger in Jemoreng Protected Forest, Sarawak. This is the first scientific discovery to describe the presence of P. c. cruciger in Sarawak in detail. According to Nijman et al. (2020), it is a rare primate, accounting for < 5% of the primate historical distribution. Based on the International Union for Conservation of Nature’s Red ist category, P. c. cruciger has been classified as a critically endangered subspecies (IUCN 2024). Its population size has decreased by ~ 80% over the past 30 years (Nijman et al. 2020). The increasing land conversion into oil palm plantations has become a major threat to the survival of P. c. cruciger (Nijman et al. 2020). P. c. cruciger is one of the most neglected transboundary primates in Sarawak and Kalimantan (Md-Zain 2019, Ampeng et al. 2024).

Considering the possible extinction of this subspecies, it is essential to take immediate action and adopt relevant measures to protect its survival. Furthermore, there is a lack of research regarding this subspecies, such as its feeding ecology and habitat utilisation (Santoso et al. 2023b, Ampeng et al. 2024). There are insufficient data to fully comprehend this species’ ecology in its native environment (Nijman et al. 2020). A thorough review of how this species is affected by its ecological communities, habitat change and revising conservation plans requires a prior understanding of their diets in the wild (Osman et al. 2020, Abdullah-Fauzi et al. 2022, Mohd-Radzi et al. 2022, Osman et al. 2022). Primate diets of P. c. cruciger have been described from the Kalimantan population (Santoso et al. 2023a, Santoso et al. 2023b) and comprehensive feeding ecology information in Sarawak is available for its sister taxon, P. c. chrysomelas, in Samunsam Wildlife Sanctuary (Ampeng 2007, Ampeng and Md-Zain 2007, Ampeng and Md-Zain 2012). At present, dietary studies of primates can be conducted using a DNA metabarcoding approach as an alternative to direct ecological observation. This next-generation sequencing (NGS) technique that utilises a metabarcoding diet approach is suitable for studying the diet of the rarely sighted P. c. cruciger. An advantage of the DNA metabarcoding approach is that it identifies multiple species from a single DNA marker using degraded samples, such as faeces (Khairulmunir et al. 2023, Gani et al. 2024a, Gani et al. 2024b). As it is rare to encounter the endangered P. c. cruciger in the wild, obtaining faecal samples to evaluate its diet is necessary and beneficial.

The first sighting of P. c. cruciger in Jemoreng Protected Forest (JPF), Sarawak (2°42′00′′N, 111°39′00′′E) was made during scientific surveys conducted by the Forest Department Sarawak using a camera-trap method (Fig. 1) (Ampeng et al. 2024). During this survey, only a single faecal sample of P. c. cruciger was successfully obtained from the study site. Total genomic DNA was extracted using the QIAamp PowerFecal Pro DNA Kit according to the manufacturer’s protocol. The extraction produced high-quality DNA and its concentration was quantified using a Hercuvan Nucleic Acid Analyzer. Genetic identification of the faecal sample was performed using D-loop region sequences (Meyer et al. 2011) (Table 1) and species confirmation was performed via GenBank BLASTN (pairwise distance: 95.1% similarity with P. chrysomelas). Genomic DNA product of P. c. cruciger was sent to Apical Scientific Sdn Bhd for further NGS processing. Amplicon sequencing of the diet was performed using locus-specific primers of the trnL gene (P6 loop region) with overhang adapters (Taberlet et al. 2007) (Table 1). To accomplish library amplification, KOD-Multi & Epi-® (Toyobo) was utilised. Dual indices were affixed to the amplicon PCR using the Illumina Nextera XT Index Kit version 2, in compliance with the manufacturer’s standard protocol. The quality of the libraries was assessed using an Agilent Bioanalyzer 2100 System with an Agilent DNA 1000 Kit and via fluorometric quantification using Helixyte Greenä Quantifying Reagent. The library was normalised and pooled following the protocol recommended by Illumina and the MiSeq platform was used to perform a 150-paired end sequencing. The raw FASTQ data were filtered and assessed using fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). R Studio version 2023.09.1 with DADA2 packages analysed the obtained NGS data. All amplicon sequence variants (ASV) data were generated by filtering, denoising, merging and deleting chimeras using DADA2 (Callahan et al. 2016). The obtained ASV data were imported and used to perform dietary characterisation analysis in R Studio.

Figure 1.  

Tricolour Langur of Jemoreng Protected Forest captured using a camera-trap method.

Amplicon sequencing of the trnL gene from a single individual of P. c. cruciger successfully generated 87,473 raw reads, followed by filtered (85,116), denoised (85,095) and merged (83,816) data reads. The final non-chimeric sequences consisted of 82,488 reads, which resulted in 11 ASVs for the diet profiling analyses. From the total non-chimeric reads acquired, 6.62% of the sequences were not assigned to any taxonomy classification. From the 11 acquired ASVs, one phylum, four classes, four orders, four families, three genera and three species were identified. Streptophyta is the only identified phylum in the P. c. cruciger faecal sample. Fig. 2 shows the sunburst chart of the taxonomic composition and the relative abundance of the P. c. cruciger faecal sample at the family, genus and species levels. Menispermaceae (85.22%) is the most abundant at the family level, followed by Polygalaceae (8.01%), Urticaceae (0.12%) and Sapindaceae (0.03%). Meanwhile, the most abundant at the genus and species level, with 85.22%, are Fibraurea and Fibraurea tinctoria. Other identified genera included Poikilospermum (0.12%) and Litchi (0.03%), while other identified species were Poikilospermum suaveolens (0.12%) and Litchi chinensis (0.03%).

Figure 2.  

Sunburst taxonomy on the diet of P. c. cruciger from the Jemoreng Protected Forest.

Overall, there were three plant species which were successfully identified in their diet, based on the NGS approach: F. tinctoria (akar kuning), P. suaveolens (akar jangkang) and L. chinensis (lychee). Meanwhile, 14.63% were not identified at the species level. F. tinctoria, from the family Menispermaceae, is typically found in lowland forests — either primary, secondary or disturbed — at altitudes up to 1,200 m a.s.l. (MyBIS 2024). In Sarawak, this plant species is frequently found in peat swamp forests, including JPF, where the faecal sample of P. c. cruciger was collected. In addition, F. tinctoria was discovered in the diet of the endemic Bornean Maroon langur, Presbytis rubicunda (Ehlers-Smith et al. 2013) at Sabangau, Kalimantan. However, in contrast to P. c. cruciger, F. tinctoria was not the main preference for P. rubicunda and only 1.6% of their mean monthly feeding time was utilised to consume this plant. Meanwhile, P. suaveolens was discovered to be a part of the diet of another Presbytis species, P. fredericae, although in low abundance (Suryana 2010), indicating that F. tinctoria is a common plant species consumed by the genus Presbytis.

Reportedly, P. c. cruciger of Danau Sentarum National Park (West Kalimantan) feeds on 27 types of plant species (Santoso et al. 2023b). Gita susu (Willughbeia coriacea), Merepat (unidentified) and Karet (Hevea brasiliensis) were their main diets. Concerning feeding composition, Santoso et al. (2023b) described that P. c. cruciger mainly preferred leaves (50%), followed by fruits (30%) and seeds (20%). Furthermore, P. c. cruciger preferred feeding from trees (89%) and lianas (11%) (Santoso et al. 2023b). Plant species F. tinctoria, P. suaveolens and L. chinensis have small fruits. Previous studies have also reported that P. c. cruciger usually consumes small- to medium-sized fruits with a diameter of 0.5–5 cm (Santoso et al. 2023b). In another study, 10 plant species were identified as the primary food source of P. c. cruciger, including Grewia paniculata, Diospyros sp. and Pternandra galeata (Santoso et al. 2023a). However, its sister taxon, P. c. chrysomelas, from Samunsam Wildlife Sanctuary (Western Sarawak), consumes Dipterocarpaceae, followed by Anacardiaceae and Myrtaceae (Ampeng 2007). When there are several diet options, the animal’s preference for a specific food is determined by its palatability (‌Prescott 2006, Kirschner et al. 2014). A few factors may contribute to the palatability for a primate, such as food availability, food abundance and distribution, nutritional content and energy (Solanki et al. 2008, Grueter et al. 2009, Osman et al. 2020, Strier 2021).

The preliminary dietary intake data of the rare P. c. cruciger is important for their conservation strategies, particularly for plant species that are highly consumed by this primate. Their population has been decreasing for the past few years, with an unknown population size at present. Maludam National Park was the only known record for the existing distribution of P. c. cruciger in Sarawak (Nijman et al. 2020). Thus, the recent discovery of P. c. cruciger in JPF has provided research opportunities for comprehensive feeding ecological studies. Knowing the importance of certain food resources, such as how the available food suits the nutritional demands, primates’ flexible diets, value of fallback foods and nutritional needs of the various species, will be essential for conservation strategy planning (Chapman et al. 2012).

In conclusion, these preliminary findings identified three plant species consumed by P. c. cruciger: F. tinctoria (Menispermaceae), P. suaveolens (Urticaceae) and L. chinensis (Sapindaceae). Acquiring knowledge and understanding their diets is essential for the conservation management purposes of P. c. cruciger and their preferred consumed plant where its population inhabit. Further research studies on its behavioural ecology and genetic aspects are necessary to comprehend the rare and critically endangered P. c. cruciger in Sarawak.

We thank the Forest Department Sarawak especially Hamden Mohammad, Happysupina Sait, Madeline George Pau, Syafiani Osman, Emy Ritta Jinggong, Roslan Borhan, Ishak Hassim, Mohammad Fardy Abdul Rahman, Azahari Omar, Dayang Nuriza Abg Abdillah, other staff and field assistants (Tony Neyam, Mohd Zairol Abdullah, Asui Kion, Romzi Kadir) for their support. We thank Sarawak Forest Department for allowing us to use faecal samples for NGS metabarcode analyses. The field survey project is a Sarawak government project with permit No. (39) JHS/CCD/400-1/1/102/JLD.1 and has been funded through allocations B210 (State Government) and P23 (Federal Government). The primate research permits are No. SFC.810-4/6/1(2023)-065 and (43) JHS/600-9/2/102/Jld.2. The authors acknowledge The National Conservation Trust Fund for Natural Resources (NCTF), ST-2022-027, funded by the Ministry of Natural Resources and Environmental Sustainability, Malaysia and TNB Research Sdn. Bhd., for funding the present work through Grant ST-2021-017 registered at Universiti Kebangsaan Malaysia for molecular genetics work. We appreciate the help of the reviewers, who made valuable suggestions for improving the manuscript.

State Government, Federal Government, The National Conservation Trust Fund for Natural Resources (NCTF), TNB Research Sdn. Bhd.

B210 (State Government) and P23 (Federal Government), UKM ST-2022-027, ST 2021-017

Forest Department Sarawak and Universiti Kebangsaan Malaysia

RCTT wrote the manuscript: AA, JL, BMMZ Conceptualisation; AA, BD, MLA conducted field sampling; RCTT conducted laboratory work; RCTT conducted all the data analyses; AA, BMMZ critically revised the intellectual content; AA, JL, BMMZ Funding acquisition and resource. All authors read and approved the final version of the manuscript.