Conservation translocation and reintroduction for the purpose of repopulating and reinforcing extirpated or depleted populations has been recognised as an important conservation tool, particularly for gibbon conservation in the immediate future. Feasibility assessments involving multiple factors, including taxonomic and genetic assessment of rescued and captive gibbons, are imperative prior to translocation and reintroduction programmes. In this study, we attempt to determine the subspecies and origin of captive Hylobates lar, White-handed gibbons, from Peninsular Malaysia to assist in future translocation and reintroduction programmes. A total of 12 captive and rescued H. lar samples were analysed using the control region segment of mitochondrial DNA. Sequence analyses and phylogenetic trees constructed using neighbour-joining, maximum likelihood, Bayesian inference, and network methods congruently differentiate all 12 captive individuals used in this study from other H. lar subspecies suggesting that these individuals belong to the H. lar lar subspecies. In addition, two populations of H. l. lar were observed: (1) a southern population consisting of all 12 individuals from Peninsular Malaysia, and (2) a possible northern population represented by three individuals (from previous studies), which might have originated from the region between the Isthmus of Kra, Surat Thani-Krabi depression, and Kangar-Pattani. Our findings suggest that the complete control region segment can be used to determine the subspecies and origin of captive H. lar.

Control region, mitochondrial DNA, northern and southern lar populations, phylogenetic relationships, subspecies determination

Small apes (family Hylobatidae), also known as lesser apes, consist of 20 species of gibbons inhabiting Southeast Asia which are grouped into four extant genera: Hylobates, Hoolock, Nomascus, and Symphalangus. Within the genus Hylobates, nine species are currently recognised (Roos 2016; IUCN 2021); Hylobates abbotti Kloss, 1929, Hylobates agilis F. Cuvier, 1821, Hylobates albibarbis Lyon, 1911, Hylobates funereus I. Geoffroy, 1850, Hylobates klossii (Miller, 1903), Hylobates lar (Linneaus, 1771), Hylobates moloch (Audebert, 1798), Hylobates muelleri Martin, 1841 and Hylobates pileatus (Gray, 1861); two species exist in Peninsular Malaysia namely H. agilis and H. lar. In Peninsular Malaysia, H. lar is distributed throughout except for a narrow region between Perak River (State of Perak) and Muda River (State of Kedah) that is inhabited by the congener, H. agilis (Brockelman & Geissmann 2020). Both species are categorised as ‘Endangered’ by the IUCN Red List of Threatened Species (Brockelman and Geissmann 2020; Geissmann et al. 2020) and are ‘Totally Protected’ under the Wildlife Conservation Act 2010 enforced in Peninsular Malaysia. Illegal hunting for the food and pet trade as well as habitat loss due to anthropogenic activities (forest clearing for development and agriculture) have been identified as the major causes of the decline of more than 50% of H. lar populations in the wild across its range (Brockelman and Geissmann 2020).

Large numbers of captive gibbons kept in zoological parks (including zoos and rescue centers) are individuals rescued from the illegal pet trade, private collectors, and plantations as their habitats are cleared (Cheyne 2009; Nijman et al. 2009). Due to the threat faced by in-situ populations, conservation translocation and reintroduction of ex-situ populations (of rescued and captive individuals) for the purpose of repopulating and reinforcing extirpated or depleted populations has been recognised as an important conservation tool, particularly for gibbon conservation in the immediate future (Cheyne 2009; IUCN/SSC 2013; Campbell et al. 2015). However, before such conservation actions are taken, feasibility assessments involving multiple factors including taxonomic and genetic assessment of rescued and captive gibbons are imperative prior to any translocation and reintroduction programmes (Campbell et al. 2015).

To assess the taxonomic and genetic variation of gibbons, several molecular taxonomy studies have been conducted. However, most systematic studies on gibbons have focused mainly on interspecific variation (Chan et al. 2010, 2012, 2013; Israfil et al. 2011; Veeramah et al. 2015; Matsudaira and Ishida 2021) while only a few have investigated intraspecific variation (Andayani et al. 2001; Woodruff et al. 2005; Aifat and Md-Zain 2021). In particular, the taxonomic status of H. lar subspecies requires further examination as they are based on minor variations in body colour and fur polychromatism (Woodruff et al. 2005; Brockelman and Geissmann 2020). According to Roos et al. (2014), five subspecies of H. lar are currently recognised: H. l. lar (Linnaeus, 1771), H. l. carpenteri Groves, 1968, H. l. entelloides I. Geoffroy Saint-Hilaire, 1842, H. l. vestitus Miller, 1942, and H. l. yunnanensis Ma & Y. Wang, 1986. In this study, we employ the control region (CR) gene segment, a more variable gene segment of the mitochondrial DNA (mtDNA) (Roos and Geissmann 2001; Woodruff et al. 2005; Whittaker et al. 2007; Rovie-Ryan et al. 2014), to assess the taxonomic and genetic variation of white-handed gibbons in captivity.

A total of 1030-bp of the complete CR of mtDNA was successfully obtained from all 12 samples used in this study using the newly designed pairs of oligonucleotides. The CR segment of mtDNA is the most variable region and has been recommended as the appropriate segment to be used to infer gibbon phylogeny (Roos and Geissmann 2001; Woodruff et al. 2005; Whittaker et al. 2007; Matsudaira and Ishida 2010; Aifat and Md-Zain 2021). However, due to the lack of complete CR sequences of Hylobates in GenBank, a total of 527-bp of aligned dataset were analysed from 100 sequences representing all species of Hylobates except H. funereus. Our initial analysis of the complete CR sequence of H. lar (aligned sequence length of 1110-bp) showed an additional 59 VS and 34 PIS which may provide more informative data for the taxonomic and genetic assessment of H. lar. Therefore, we recommend the continued use of the complete CR segment in future studies of gibbon phylogeny and taxonomy.

The phylogenetic relationships among Hylobates remain unresolved. Firstly, we observed H. pileatus as the basal species of Hylobates (although with low support), a result that is consistent with previous studies (Israfil et al. 2011; Chan et al. 2013; Matsudaira and Ishida 2021). Secondly, the relationships among the Sundaic species group (abbotti, agilis, albibarbis, klossii, moloch, and muelleri) remains conflated particularly with regard to the basal species of this group (Thinh et al. 2010; Chan et al. 2013; Matsudaira and Ishida 2021). In this study, the Bornean species group (abbotti and muelleri) diverged first followed by the Indonesian species group (agilis, klossii, and moloch). The clustering of H. albibarbis within the H. agilis clade is expected because the subspecies albibarbis was initially classified as a subspecies of H. agilis (Thinh et al. 2010; Matsudaira and Ishida 2021).

The phylogenetic positioning of H. lar within the genus Hylobates concurs with findings from previous studies (Israfil et al. 2011; Chan et al. 2013; Matsudaira and Ishida 2021). Our phylogenetic and network analysis showed that H. l. vestitus from Sumatra diverged first (Thinh et al. 2010; Aifat and Md-Zain 2021; Matsudaira and Ishida 2021) before the proto-H. lar bifurcated to form the H. l. lar cHylobateslade and the possible Indochinese subspecies clade (entelloides, carpenteri, and yunnanensis). However, the Indochinese subspecies clade did not show any obvious subspecies groupings. Note that most of the sequences of the Indochinese subspecies clade were obtained from zoo individuals of unknown origin and locality (Suppl. material 1) except for sequences by Matsudaira et al. (2013) (AB720992 - AB721001) which originated from individuals from Khao Yai National Park, Thailand representing the subspecies H. l. entelloides. Thus, we deduce these following scenarios to explain the condition: (1) Indochinese lar gibbons are represented by only one subspecies (entelloides) instead of three, (2) sequences of lar gibbons from GenBank of unknown locality were derived from only one subspecies, H. l. entelloides, and (3) the CR segment is not powerful enough to differentiate among the Indochinese subspecies. In view of the lack of information regarding the origin of the zoo animals as well as the lack of reference samples of known locality, these proposed scenarios must be regarded as hypothetical and preliminary. Future studies should therefore use individuals of known localities to correctly assign captive and rescued gibbons to their geographical provenance and origin.

Finally, our findings revealed that all 12 captive individuals used in this study belong to the H. l. lar subspecies. Aifat and Md-Zain (2021) used partial cytochrome b gene segment of the mtDNA to successfully identify the subspecies of captive H. lar in Peninsular Malaysia but suggested that the CR segment be used in further studies on H. lar. The CR segment has been used in forensic investigations of various wildlife species to identify the subspecies and geographical origins of rescued or confiscated animals (Iyengar 2014). Interestingly, our results suggest the occurrence of two distinct populations of H. l. lar, as demarcated by two transversion mutations at np 165 and 259: (1) a southern population consisting of all 12 individuals from Peninsular Malaysia, and (2) a possible northern population of H. l. lar represented by three individuals from previous studies (AF311723, LC548024, and LC548028). These transversion mutations may be due to the prolonged isolation between the postulated southern and northern populations, which are separated by the presence of H. agilis in the narrow region between the Perak River and Muda River (Figure 1). Similar findings have been observed in other non-human primates, such as Macaca (Rovie-Ryan et al. 2021). Thus, these mutation sites should be used as genetic markers for future taxonomic and genetic assessments involving lar gibbons. The southern H. l. lar population likely consists of all populations south of the Kangar-Pattani line (Fig. 1) and including the entirety of Peninsular Malaysia. In contrast, the northern population may exist in regions between the Isthmus of Kra and the Kangar-Pattani line. Several previous studies have demonstrated the role of the Isthmus of Kra, the Surat Thani-Krabi depression, and the Kangar-Pattani line as zoogeographical boundaries between Sundaland and Indochina (Baltzer et al. 2007; Woodruff 2010; Hughes et al. 2011; Rovie-Ryan et al. 2013; Abdul-Latiff et al. 2014).

Overall, our findings support the importance of conducting taxonomic and genetic assessments prior to any gibbon translocations and/or reintroductions. The distinguishable differences between the postulated northern and southern H. l. lar populations warrant their treatment as separate management units (MUs), a component within the Evolutionary Significant Unit (ESU) (Moritz 1994). Although treated as separate MUs, under the circumstance that the remnant populations of lar are showing signs of inbreeding depression or increased fragmentation, mixing (translocation) between MUs are permittable (Moritz 1999).

In summary, we conclude that using the CR segment of the mtDNA, we could taxonomically distinguish H. l. lar from the other H. lar subspecies, an important result for future translocation and reintroduction programs of rescued and captive gibbons in Peninsular Malaysia. Nevertheless, the use of nuclear DNA data for taxonomic and genetic assessments of captive and rescued gibbons should also be considered, especially for individuals of suspected hybrid origin. Further studies are currently on-going by DWNP (as the authority of wildlife conservation and management) to screen all captive lar gibbons in Peninsular Malaysia as well as to collect reference samples from the wild.

We wish to thank the Director General and Director of the Ex-situ Conservation Division of DWNP for the support and permission to conduct this study. Special thanks to the following personnel for their assistance during sampling: Dr David Magintan, Dr Zubaidah Kamarudin, Dr Siti Suzana Selamat, and the Primate Team of the NWRC of DWNP. The project was funded by the Government of Malaysia under the 12^th Malaysia Plan Project: Strengthening Wildlife Forensics, Ex-Situ Conservation and Biobanking - Phase 2 (Project Code: P23071000810008) lead by JJRR. We also thank UNIMAS for the Publication Support Fee. MG and JJRR share the first authorship of the manuscript.