javascript:void(0)Introduction
The illegal wildlife trade is a major global conservation issue that
threatens the future survival of many species, including all eight
pangolin species. Pangolins, or scaly anteaters (Order: Pholidota), are
typically solitary, nocturnal mammals, found across Asia and Africa.
Pangolins have been described as the world’s most trafficked mammal
group (Aisher 2016), driven by demand for their scales and meat through
the illegal wildlife trade. Combating pangolin poaching is challenging
due to ease of capture, the secretive nature of this lucrative trade,
associated corruption (Anagnostou and Doberstein 2022) and difficulties
in monitoring and protecting pangolins in remote habitats.
Four Asian pangolin species are currently recognised: the Sunda Pangolin
(Manis javanica ), Philippine Pangolin (Manis culionensis ),
Chinese Pangolin (Manis pentadactyla ) and Indian Pangolin
(Manis crassicaudata ). Genetic data have recently suggested the
existence of a fifth Asian pangolin species (tentatively namedManis mysteria ), however, these data were derived from seized
pangolin material, so the geographic origin is unknown (Hu et al. 2020b; Gu et al. 2023). Two species of pangolin occur in Nepal,
the Chinese pangolin and the Indian pangolin (Khatiwada et al. 2020). Distribution records suggest that Chinese pangolins are
distributed across eastern, central and mid-western Nepal at elevations
of up to 2,000 m while Indian pangolins are found in Nepal’s western
regions, at lower elevations (below 500 m) (Baral and Shah 2008; Jnawaliet al. 2011; Sharma et al. 2020; Figure 1). There may be
some overlap between the ranges of the two species, though this has not
been confirmed (Khatiwada et al. 2020). Nepal, and especially
eastern Nepal (Ghimire et al. 2020) has long been considered a
major hotspot for pangolin poaching and trafficking (Thapa et al. 2014; Katuwal et al. 2015).
Given the threats faced by pangolins, there is a need to develop
efficient survey and monitoring methods to determine species status,
identify priority populations and evaluate the impact of conservation
interventions on populations. Pangolins are typically rare, cryptic
creatures that are difficult to survey and monitor. Willcox et
al. (2019) provides an overview of survey methods for pangolins,
including burrow counts, nocturnal and diurnal surveys, camera trapping,
community interviews, telemetry, use of detection dogs and molecular
techniques. The authors discuss the effectiveness of combinations of the
above listed methods, noting the potential for molecular techniques to
confirm species identification from scats (faecal samples), where, for
example, scats are used to confirm pangolin presence. Willcox et
al. (2019) also lament that there is limited genetic material available
from pangolins of known geographic origin. This is especially true of
wild pangolin populations and, along with low coverage across their
large geographic range, limits capacity to infer the origin of seized
pangolin or pangolin material using genetic analyses. Collection of
samples of known origin would facilitate the establishment of a
reference sample database that could assist with increasing our
understanding of pangolin population structure while also facilitating
genetic tracing.
Calls for research to generate knowledge regarding
pangolins, to inform policy makers and law enforcement
agencies, include the need to identify the geographical sources of
trafficked pangolins, at national and local scales (Pietersen and
Challender 2020). Accurate, georeferenced DNA databases are required to
inform an understanding of trafficking dynamics and origins of
trafficked individuals, and to guide conservation action and policy.
Molecular methods that identify genetic differences between populations
in different geographic regions can be used to create large genetic
databases that characterise populations across a species’ range (Wasseret al. 2004; Ogden et al. 2009). Such an approach was
reported by Wasser et al. (2007) for elephants, and by Ghobrialet al. (2010) for chimpanzees and can be applied to pangolins.
For example, studies of mitochondrial and nuclear DNA by Gaubertet al. (2016) revealed six geographic lineages of the African
common pangolin (Manis tricuspis ). This information could help
track the global trade of this species at a sub-regional scale.
Similarly, using samples from known origin (sampling of wild pangolins),
Nash et al. (2018) identified three main clusters of Sunda
pangolin across Indonesia and subsequently used that information (and
DNA from seized pangolin material) to infer possible trade routes.
The establishment of a georeferenced DNA database for pangolins in Nepal
relies upon sampling of wild populations, which can be challenging for
rare and cryptic species, especially where field locations are remote
and difficult to access. Non-invasive genetic sampling of scats is now a
commonly used method to obtain DNA from cryptic, threatened species for
population genetic investigations and has many advantages over invasive
sampling strategies as it allows for wide landscape surveys to be
conducted by non-experts e.g., natural resource managers or citizen
scientists. Non-invasive genetic sampling can also be used for
opportunistic or targeted sampling, and can be far more cost effective
and ethical, as DNA can be collected without the need of capturing or
even observing a target. Scat collection is therefore a good option for
genetic studies of pangolin, which are elusive and highly threatened
creatures. If scat can be preserved without affecting the presence or
quality of the DNA, sample banks can be built up over time from various
locations.
Pangolin scat DNA can be a valuable resource which can be used to
improve our understanding and knowledge about these vulnerable species.
DNA isolated from scats can be used to generate unique DNA profiles,
which allows for individual identification. Genetic data can provide
insights into the genetic diversity within populations, whilst
genotyping individuals from different geographic locations, can be used
to infer differentiation, gene flow between populations and to monitor
population trends, all of which are critical for conservation management
and prioritising populations for protection. Extensive DNA profiling can
allow the characterisation of pangolin populations which can assist
forensic investigations allowing the geographic origin and / or source
population of confiscated pangolin products to be identified.
Pangolin scats largely consist of grit (~50%) and
undigested insect matter (~30%) (Karawita et al. 2020; Mahmood et al. 2021) and contain DNA from numerous sources,
including from the animal itself (target DNA in this context), its
intestinal microbiome, pathogens, parasites and plant or animal species
it has ingested. Target DNA may therefore represent a limited proportion
of the total DNA that is obtained from a scat sample and may be present
in low amounts. Further to this, Pangolin DNA, isolated from scats is
likely to be degraded due to exposure to environmental factors such as
sunlight, moisture / precipitation and microbial activity. DNA
degradation, low DNA yield and contamination can limit the use of scats
for genetic studies. Despite these challenges, careful selection and
optimisation of methods for sample collection and genetic analysis can
produce reliable genetic data (e.g. Piggott and Taylor 2003; Luikart et
al. 2008; Wedrowicz et al. 2013).
In this study, we used DNA isolated from pangolin scats, using both
surface swabs and whole scat material to generate genetic sequence
suitable for use in establishing a georeferenced DNA database for wild
pangolin populations in Nepal. Such data will be especially useful for
mapping the geographic extent of the species and its populations,
providing a reference against which genetic evidence for investigations
and monitoring of crimes such as poaching and illicit wildlife trade can
be compared. This pilot study aimed to determine whether pangolin DNA of
sufficient quality and quantity to be useful for genetic analyses can be
obtained from pangolin scat samples.