Charlotte E. Hacker Science Reviews - Biology, 2023, 2(3), 1-7
Decoding the DNA of scat and the application of ge-
netic methodologies to understanding carnivore diet
Charlotte E. Hacker, PhD
The Snow Leopard Conservancy; Sonoma, California, USA;
Received July 17, 2023. Revised July 25, 2023. Published online August 28, 2023.
Abstract: Carnivore species are vital to ecosystem function and maintenance. One key component to
understanding carnivore ecology and the most effective means of management is knowledge of dietary resource
use. Traditional methods used to study carnivore diet, such as microhistology, have several technical and
logistical shortcomings. These have hindered the quality and quantity of data that shape understanding of how
carnivores exploit prey. Advances in genetic methodologies and their application to wildlife biology has
transformed the manner in which information about species can be gained. DNA metabarcoding is one such
example. With this approach, genetic sequences present in scat can be determined via next-generation
sequencing and matched to reference databases, revealing the carnivore that deposited the scat and the prey it
consumed. DNA metabarcoding has the ability to overcome many of the previous challenges associated with
dietary analysis and works to advance and inform current knowledge surrounding carnivore ecology, predator-
prey relationships, conflicts between carnivores and humans, and potential adaptability to large-scale landscape
shifts. Its use has provided novel insights on numerous carnivore species to help inform research priorities and
wildlife policies, including those living in unique fragile environments such as the Qinghai-Tibetan Plateau of
China. The continued development and increased capacity of molecular dietary analysis via DNA
metabarcoding has the promise to grossly improve carnivore conservation management strategies on a global
Keywords: Carnivore, Conservation Genetics, DNA Metabarcoding, Molecular Dietary Analysis, Next-
Generation Sequencing, Predator
Carnivores play key roles in the functional
maintenance of the ecosystems they occupy. They
influence herbivore populations which in turn con-
trols vegetation (Ripple et al., 2014), impact disease
dynamics and parasite transmission (Terborgh &
Estes, 2010), and provide carrion for scavengers
(Wilmers et al., 2003). Successful habitat conserva-
tion and management may hinge on the holistic un-
derstanding of carnivore species in a given area,
and necessitates the obtainment of information on
their ecology, natural history, and diet.
The importance of understanding carnivore diet
Knowledge of carnivore diet can inform the
accessibility and availability of prey, the degree of
competition between different carnivore species
and how resources are partitioned, the extent of
livestock loss and its potential economic impact on
human livelihoods, as well as the adaptive potential
of a species and how it may respond to large-scale
environmental changes (Symondson et al. 2002;
Foster et al., 2013; Hacker et al., 2022).
Traditional methods used for carnivore diet
The dominant method for understanding car-
nivore diet has historically been microhistology.
With this approach, a sample, typically scat, is non-
invasively collected in the habitat where the carni-
vore of interest is found. The scat sample is then
dried, and digested hairs or bones are extracted for
Charlotte E. Hacker Science Reviews - Biology, 2023, 2(3), 1-7
comparison to the reference material of already-
known species (Pompanon et al., 2012). Another
method, albeit most commonly used to assess live-
stock predation rather than native prey, is the inter-
viewing of local community members sharing land-
scapes with carnivores (Meena et al., 2011; Oli et al.,
1994). Carnivores outfitted with global positioning
systems (GPS) have also been used as a way to find
kill sites and subsequently identify prey species (Ba-
con et al., 2011; Johansson et al., 2015), as has the ex-
amination of carnivore stomach contents (Balesrieri
et al., 2011).
Limitations and disadvantages of traditional
methods of analysis
Each of the above methods has drawbacks
that limit the ability to accumulate large amounts of
accurate data. Microhistology is a tedious process
that can be time intensive and subject to misidenti-
fication errors and inter-observer bias (Pompanon
et al. 2012). Biological reference material may not be
available and the host carnivore thought to have de-
posited the scat may be erroneously identified
(Weiskopf et al., 2016). With interviews, respond-
ents may incorrectly recall predation events, misi-
dentify the carnivore responsible, are subject to so-
cial pressure and bias, and may experience fatigue
or disinterest if the interview is too long (De Vaus &
De Vaus, 2013; Fisher 1993; Oli et al., 2014). Use of
GPS coordinates to identify kill sites requires the
carnivore to be GPS collared and the kill site to be
physically accessible to researchers (Johansson et al.,
2015). Lastly, the analysis of stomach contents re-
quires either euthanasia of the animal, or depend-
ence on fresh carcasses resulting from natural and
non-natural mortality events, such as roadkill.
Novel approaches to dietary analysis
Advancements in genetic methodologies have
resulted in the application of novel approaches to
replace the methods described above. Next-genera-
tion sequencing (NGS) platforms, such as Illumina,
now allow scientists to quickly and accurately de-
termine the order of nucleotides that comprise
DNA (A, C, T, G), resulting in terabytes of data in
the form sequencing reads. Online databases such
as NCBI GenBank
( and
BOLD ( have be-
come reservoirs of uploaded genetic sequences for
species genes and genomes. These known se-
quences serve as references by which sequencing
reads obtained by NGS can be uploaded, matched,
and compared for identification purposes.
The method of DNA metabarcoding
NGS technology is leveraged to study carni-
vore diet via DNA metabarcoding (Figure 1). With
this method, researchers collect scat in the field and
then bring it to a laboratory. The DNA present in
the scat is then non-specifically extracted from the
sample and subjected to PCR (polymerase chain re-
action) with a pair of universal primers. Universal
primers are less-specific and smaller stretches of
pre-made genetic sequences that attach to target ar-
eas of the animal’s DNA. These primers then “run”
across the DNA to make copies of a specific gene
segment of interest via a series of pre-programmed
cycles in a thermocycler. The selection of the seg-
ment of DNA to be amplified and sequenced is im-
portant. It must be conserved enough so that all
possible species possess it, but different enough in
its nucleotide sequence to tell them apart (Valentini
et al., 2009). For example, mitochondrially encoded
12S ribosomal RNA (MT-RNR1) is found in all ver-
tebrate species, but may not be different, or differ-
ent enough, between all vertebrate species (Shehzad
et al., 2012).
The resulting genetic material undergoes an-
other round of PCR to attach index tags to each in-
dividual sample so that they can be multiplexed
into one tube and later “pulled out” of the mix for
analysis. The final product of pooled samples with
index tags is pipetted onto a flow cell and placed on
the sequencer. Once finished, data files of returned
sequences and other documents, such as a quality
control report, are available for retrieval and analy-
sis using bioinformatics.
Bioinformatic tools allow researchers to ana-
lyze and process complex and voluminous genetic
data in a streamlined, collaborative, and efficient
manner (Gauthier et al., 2018). Researchers load re-
turned sequences onto a computer, and use bioin-
formatic pipelines and software programs to match
returned sequences to reference sequences. Param-
eters and other predetermined metrics are used to
help ensure confidence in the determination of the
host species as well as the prey item.
There are a number of key benefits to DNA
metabarcoding, many of which overcome the limi-
tations associated with more traditional dietary
analysis approaches. DNA metabarcoding allows
for multiple samples to be processed rapidly in tan-
dem, resulting in large amounts of data in a
Science Reviews - Biology, 2023, 2(3), 1-7 Charlotte E. Hacker
relatively short period of time (Shokralla et al., 2012).
It also eliminates issues surrounding human-ob-
server bias and produces more accurate infor-
mation (Pompanon et al., 2012; Valentini et al., 2009).
Further, DNA metabarcoding requires only a small
amount of scat as starting material. Alternatively,
microhistology may necessitate collection of the en-
tire sample, which takes up more space, challenges
transportation logistics and costs, and fully re-
moves potentially important chemosensory infor-
mation from the environment.
Figure 1:
A workflow example showing the application of DNA metabarcoding to determining carnivore diet.
Using DNA metabarcoding to study carnivore diet
in China
One area where DNA metabarcoding has
been heavily leveraged to understand carnivore
diet is the Qinghai-Tibetan Plateau (QTP) of China.
The QTP is a 2.5 million km
landscape with an av-
erage altitude of 4,500 m above sea level (Zhang et
al., 2002). The relatively recent and rapid uplift of
the plateau has led to numerous ecosystems and en-
demic species found nowhere else in the world
(Chang, 1981). Carnivores on the QTP include the
Tibetan wolf (Canis lupus), Tibetan brown bear (Ur-
sus arctos pruinosus), snow leopard (Panthera uncia),
Eurasian lynx (Lynx lynx) (Xu et al., 2008), Tibetan
fox (Vulpes ferrilata), red fox (V. vulpes), Pallas’s cat
(Otocolobus manul), and Asian badger (Meles
leucurus), among others. Native prey species in-
clude blue sheep (Pseudois nayaur), Tibetan gazelle
(Procapra picticaudata), argali (Ovis ammon), white-
lipped deer (Cervus albirostris), marmot (Marmota
spp.), chukar partridge (Alectoris chukar), and pika
(Ochotona spp.), among others (Jackson, 2012; Schal-
ler et al., 1988). Pastoralism remains heavily prac-
ticed in the area by local Tibetans and a number of
livestock that may serve as food for wild carnivores
are also present. These include domestic yak (Bos
grunniens), goat (Capra aegagrus hircus), sheep (Ovis
aries), and horse (Equus caballus).
Livestock depredation causes financial bur-
dens on herders, their families, and the local econ-
omy, promotes negative attitudes towards carni-
vores, is emotionally traumatizing, can create ten-
sion between residents and agencies seeking to pro-
tect at-risk species, and can lead to retaliatory kill-
ings . Herders who retaliate may also sell body parts
to recuperate their financial losses depending on the
carnivore killed, which can contribute to the illegal
wildlife trade . Understanding the use of livestock
as a food source is needed for carnivore conserva-
tion in areas dominated by pastoralism, as it is nec-
essary for constructing tailored mitigation strate-
gies, validating herder knowledge to outside
Charlotte E. Hacker Science Reviews - Biology, 2023, 2(3), 1-7
entities, and designing assistance programs such as
livestock insurance .
In an effort to better understand the role that
different carnivore species play in livestock loss on
the QTP, DNA metabarcoding was used to deter-
mine the livestock species in 147 red fox, 25 Tibetan
fox, 153 Tibetan wolf, 191 snow leopard, and 72 Eur-
asian lynx scats collected from September 2017 to
July 2018 in Qinghai and Gansu Provinces (Hacker
et al., 2022). As expected via previous interviews
with local herders (Hacker et al., 2020), the Tibetan
wolf had the highest percentage of livestock in their
diet at 21.7% of total prey counts, followed by red
fox at 5%, snow leopard at 4.9%, Tibetan fox at 4%,
and Eurasian lynx at 3.6%. Domestic yak were most
often identified, followed by sheep, and then goat.
Interestingly, two isolated occurrences of horse and
pig (Sus scrofa) were identified as being consumed
by snow leopard, a species that is often described as
a near-specialist (Lyngdoh et al., 2014). The degree
of livestock present in the diets of smaller carni-
vores such as the red and Tibetan fox was unex-
pected, but may be attributable to scavenging or the
depredation of newborns. Unfortunately, DNA
metabarcoding is unable to reveal the life stage of
the prey identified and cannot confirm if the host
carnivore killed the species it ultimately consumed.
From these findings, mitigation measures that
work in the interest of local herders and at-risk car-
nivore species could be recommended. For example,
the dominance of yak in Tibetan wolf diet helped to
validate herder knowledge, and assisted in leverag-
ing support for yak protection practices specifically
tailored to wolf ecology. In addition, the diversity
of livestock in snow leopard diet provided insight
into the potentially opportunistic and adaptable na-
ture of the species. All herders may want to take
protective measures if a snow leopard is in the area
regardless of livestock species kept. However, the
overall low percentage of livestock in snow leopard
diet suggests that losses caused by this carnivore
are relatively uncommon.
Continued dietary work on the QTP will be
imperative given the fragile nature of its unique
ecosystems and endemic species present. Climate
change, rapid urban development, and shifts in tra-
ditional practices will continue to impact the region,
including the human livelihoods and wildlife that
reside there. Carnivores in shifting landscapes may
find themselves overlapping with previously
unencountered species that outcompete them, caus-
ing an increased pursuit of livestock. Dietary infor-
mation can be a helpful indicator of ecosystem mod-
ifications that alter the distribution and abundance
of species as well as predicting how carnivores and
prey may adapt to future changes.
Application of DNA metabarcoding for species in
other parts of the world
DNA metabarcoding has also elucidated the
feeding habits of mammalian carnivores in other ar-
eas of the world. The analysis of 96 scat samples be-
longing to Tasmanian devils (Sarcophilus harrisii) in-
troduced to Maria Island, Tasmania revealed the
first instance of domestic cat (Felis catus) (McLennan
et al., 2022). It also confirmed that the drastic de-
creases in short-tailed shearwater (Puffinus tenuiros-
tris) and little penguin (Eudyptula minor) popula-
tions were likely due to Tasmanian devils (McLen-
nan et al., 2022). Eurasian otters (Lutra lutra) were
found to have a potentially much more flexible
feeding strategy than previously known in western
China (Wang et al., 2022). The examination of red
fox scats in Scotland found a curiously high per-
centage of domestic dog (C. l. familiaris) DNA. Re-
searchers linked this finding to coprophagia, which
may help to sustain red fox populations when their
natural prey base fluctuates (Waggershauser et al.,
2022). Coyotes (C. latrans) in New York City were
expected to have human food in their diet, but DNA
metabarcoding showed that domestic chicken (Gal-
lus gallus), presumably intended for human con-
sumption, comprised nearly half of dietary counts
(Henger et al., 2022).
An evaluation of DNA metabarcoding
Despite the vast information and novel find-
ings generated by DNA metabarcoding, it remains
an intensive and sensitive procedure that is expen-
sive, requires access to advanced laboratory equip-
ment and computers, and demands rigorous train-
ing in molecular techniques and bioinformatics.
Even minor changes in laboratory practices or pro-
tocols can cause differences in data when compared
between labs. Contamination risk is also high
(Hacker et al., 2021; Pompanon et al., 2012). The mo-
lecular marker, or genetic segment, selected must be
one that can differentiate carnivores and prey down
to a level appropriate for the research question to be
answered (Hacker et al. 2021). Further, scientists
Science Reviews - Biology, 2023, 2(3), 1-7 Charlotte E. Hacker
must ensure that uploaded sequences to be used as
references are available and accurate .
Advancements in the molecular dietary anal-
ysis of carnivores will continue to move forward as
genetic methodologies improve and become more
accessible. Recent notable insights into the ecology
and behavior of various species demonstrates the
ability of such technology to shift understanding
and apply novel information to wildlife manage-
ment plans. Continued capacity in DNA metabar-
coding has the potential to improve our under-
standing of many more traditionally difficult-to-
study carnivores, ultimately enhancing the conser-
vation outcomes that impact them and the humans
they share landscapes with.
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Conflict of Interest
No conflicts of interest to declare.