javascript:void(0)Discussion
Techniques described in this study are useful to genetically characterise wild populations over large spatial scales, and therefore allow the identify of locations from which seized/traffic individuals have come, supporting antipoaching measures. Current and future genetic technologies will be crucial in the combat against illicit wildlife trade and help protect endangered species, including pangolins (Nash et al. 2018; Hu et al. 2020a; Heighton et al. 2023; Priyambada et al. 2023). In this study we have demonstrated that DNA, isolated from scats, can be used to identify the geographic origins of specimens. Such information could be pivotal in identifying poaching hotspots and may lead to more effective law enforcement efforts in areas where illegal trade is most prevalent. Genetic profiling can also be used to identify individuals which is useful for wildlife forensic investigations as it can be used to link seized wildlife products to specific crimes and individuals (Wasser et al. 2018; Wasser et al. 2022). The use of genetic identification in anti-poaching efforts can serve as a deterrent, discouraging poachers, whilst also providing an opportunity to increase public awareness and education about the consequences of wildlife trade.
Determining the geographic origin of threatened species is not possible without knowledge of the genetic differences which exist within a species across a landscape. Using non-invasive genetic sampling is a valuable tool as it allows for the collection of genetic material from species without harming or disturbing them. It also provides a mechanism to increase sampling efforts by engaging communities and other non-specialists in the sample collection process. Isolating high-quality DNA from scats can be challenging due to the presence of inhibitors, degradation and low DNA concentration. This study demonstrates that DNA useful for genetic analysis of pangolin can be successfully sourced from scat samples, even where those samples have been previously frozen. Scats appearing in good condition (fresher scats) at the time of collection provided better quality DNA than samples that appeared in poor condition, which is consistent with other genetic studies of mammals that have used DNA isolated from scats (e.g. Piggott 2004; Schultz et al. 2018). It is also important to note, however, that viable DNA was also obtained from some scats appearing in poor condition and attempting analysis from such scats may be important where samples are from underrepresented regions. Compared to the initial trial results reported here, further method optimisation may increase DNA quantity and quality that can be obtained from pangolin scats. For example, this trial used a very small amount of scat starting material. Increasing the amount of scat that is washed may raise the amount of pangolin DNA obtained. Different DNA isolation kits can also have a substantial impact on DNA quantity and quality (e.g. Wedrowicz et al. 2019), therefore trialling various methods may be useful to maximise the quantity and quality of DNA that can be obtained from pangolin scats.
The ability to obtain pangolin DNA from their scats has also been demonstrated by Priyambada et al. (2023) who used faecal DNA isolates to sequence a portion of the mtDNA cytB gene and to genotype samples using 20 microsatellites, highlighting eight microsatellite loci with high amplification success (91 – 100%) and low rates of genotyping error (< 6%). Confidently inferring the location of origin for seized pangolins or pangolin products relies heavily on having access to reference data from individuals sampled from across their distribution. Widespread sampling of live, wild individuals would be very difficult to achieve and also raises ethical concerns. DNA sourced non-invasively from pangolin scats offers the opportunity to be able to produce such a database, by allowing large numbers of samples to be sourced without having to interfere with live animals. A coordinated program for collecting pangolin scat samples across their range for the purpose of building a DNA database would be a significant benefit to pangolin conservation efforts.
Both mtDNA and microsatellites may be useful markers from which to obtain data for building a genetic map of Chinese pangolin across their range. Both are PCR based markers which is important for maximising the chance of obtaining data from low quality DNA sources such as confiscated scales (Hsieh et al. 2011). Compared to mtDNA, nuclear DNA markers such as microsatellites or SNPs may provide more fine scale resolution of population structure and hence a better ability to pinpoint the origins of seized pangolins. Microsatellite genotyping using next generation sequencing may be a useful way by which to increase genotyping success from low quality sample sources and decrease costs (De Barba et al. 2016). Due to high copy numbers per cell, mtDNA markers are more likely to successfully amplify with increasing degradation and would still indicate geographic regions of origin (although perhaps at a broader scale). Amplicon sequencing of mtDNA markers using high throughput platforms may increase sensitivity and sequencing success while also reducing costs where large numbers of samples are to be processed (Andrews et al. 2018).
Evidence for distinct lineages of Chinese pangolins has been reported previously (see Appendix, Fig. A2). Hu et al. (2020a) reported two strongly differentiated lineages of Chinese pangolin, with one group representing samples from China (Yunnan, Guangdong, Hunan, Hainan and Taiwan) and Thailand and the second group represented by samples of uncertain origin as they were obtained from seizures on the Sino-Burmese border. Hu et al. (2020a) estimated that the two lineages of Chinese pangolin diverged 130,000 years ago at the time of the most recent uplift of Tibetan plateau. Such events resulting in changes in topography and climate are important factors contributing to currently observed distributions of diversity across landscapes (He and Jiang 2014; Hu et al. 2020a).
Three subspecies of Chinese pangolin are currently recognised and include Manis pentadactyla aurita distributed across the Asian mainland and two island subspecies, Manis pentadactyla pentadactyla from Taiwan and Manis pentadactyla pusilla from Hainan, China (Sun et al. 2021). The amount of divergence between Chinese pangolins sampled in Nepal and those sampled in China was found to be significantly greater than the amount of divergence observed between subspecies from the Chinese mainland and Taiwan (Fig. 3). This was also reported by Hu et al. (2020a) who showed that Chinese pangolin, putatively from Myanmar, were substantially more divergent from both island (Taiwan and Hainan) and mainland pangolins (detected in China and Thailand) than mainland pangolins were from island pangolins (based on COI sequences; Hu et al. 2020a).
Another study utilising pangolins sampled within Nepal and the cytochrome oxidase I gene (COI) found that Chinese pangolin sampled in Nepal clustered separately from those sampled elsewhere in Asia (Shrestha et al. 2020). Chinese pangolins sampled in the Darjeeling district of northwest Bengal, India (less than 100 km east from the Taplejung region where pangolins were sampled for this study) identified four cytB haplotypes (Priyambada et al. 2023). The Priyambada et al. (2023) study utilised a different portion of the cytB gene than used in this study, so data were unable to be directly compared, however, the data presented also appear to suggest that pangolin from the Darjeeling district are also clearly distinct from Chinese pangolins sampled in China and Thailand (Priyambadaet al. 2023).
Together, these data suggest that at least one distinct lineage of Chinese pangolin is distributed from Nepal in the west across to at least Myanmar in the east (Appendix, Fig. A2). Further work is needed to clarify the distribution of this distinct lineage, whether there are additional distinct groups of Chinese Pangolin and if morphological differences between groups exist suggesting that the detected lineages may represent separate subspecies or species.
Further diversity within the mainland Chinese pangolin may be identified with more thorough sampling across this species range, suggestions of which have been previously reported. Chromosome diploid numbers of both 2n = 38 and 2n = 40 have been reported for the Chinese pangolin located in southern China (Wu et al. 2007; Nie et al. 2009). Two forms of Chinese pangolin have been reported (both occurring in Yunnan, China) which differ in scale shape and colour and are referred to as dusky and brown Chinese pangolins (Zhang and Shi 1991). Dusky Chinese pangolin have opaque, grey-black scales while the brown Chinese pangolin has transparent, yellowish-brown scales (Zhang and Shi 1991). Dusky and brown Chinese pangolins were found to have very low levels of differentiation using protein electrophoresis (Su et al. 1994) and greater levels of differentiation using mtDNA restriction enzyme digest patterns (Zhang and Shi 1991). More recent genetic studies comparing the two forms and reports of their distributions are lacking. Further work is needed to ascertain whether these observations align with one another and the divergent lineages or whether these might represent additional diversity within the Chinese pangolin population.
The value in establishing a pangolin DNA database to support conservation and to assist in detecting and solving wildlife crime is high and significant. The ability to source DNA from scats provides the means to achieve this. A comprehensive DNA database for pangolins in Nepal will allow genetic characterisation of sampled populations, allowing identification of significant populations and documentation of the distribution and amount of diversity currently present within pangolin populations. Such data would provide a valuable resource to allow the origins of seized pangolin or pangolin products to be inferred, which may aid managers in decisions about where best to focus protection efforts. Developing a genetic database for Chinese pangolin across its extensive range is a substantial undertaking that would require investment and collaboration between research groups and governments in the Asian region.