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.