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Genetic identification of carnivore scat: implicationof dietary information for human–carnivore conflictin the Annapurna Conservation Area, NepalRaj Kumar Koirala a b , Achyut Aryal c , Christophe Amiot d , Bikash Adhikari b , DibeshKarmacharya e & David Raubenheimer aa Nutritional Ecology Research Group, Institute of Natural Sciences, Massey University,Auckland, New Zealandb Institute of Forestry, Tribhuvan University, Pokhara, Nepalc Ecology & Conservation Group, Institute of Natural Sciences, Massey University,Auckland, New Zealandd Human-Wildlife Interaction Research Group, Institute of Natural Sciences, MasseyUniversity, Auckland, New Zealande Center for Molecular Dynamics-Nepal, Kathmandu, NepalPublished online: 22 Nov 2012.

To cite this article: Raj Kumar Koirala , Achyut Aryal , Christophe Amiot , Bikash Adhikari , Dibesh Karmacharya& David Raubenheimer (2012) Genetic identification of carnivore scat: implication of dietary information forhuman–carnivore conflict in the Annapurna Conservation Area, Nepal, Zoology and Ecology, 22:3-4, 137-143, DOI:10.1080/21658005.2012.744864

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Genetic identification of carnivore scat: implication of dietary information forhuman–carnivore conflict in the Annapurna Conservation Area, Nepal

Raj Kumar Koiralaa,b*, Achyut Aryalc, Christophe Amiotd, Bikash Adhikarib, Dibesh Karmacharyae

and David Raubenheimera

aNutritional Ecology Research Group, Institute of Natural Sciences, Massey University, Auckland, New Zealand; bInstitute ofForestry, Tribhuvan University, Pokhara, Nepal; cEcology & Conservation Group, Institute of Natural Sciences, Massey University,Auckland, New Zealand; dHuman-Wildlife Interaction Research Group, Institute of Natural Sciences, Massey University, Auckland,New Zealand; eCenter for Molecular Dynamics-Nepal, Kathmandu, Nepal

(Received 13 August 2012; final version received 23 October 2012)

Human-leopard conflict exists throughout the distribution range of leopards. The management of such interactions withhumans is a high priority for the Nepalese authorities. To gain information on the extent of the human-leopard conflictin the region, we collected carnivore scat over an area of approximately 400 km2 in the lower belt of the AnnapurnaConservation Area, Nepal. We used species-specific polymerase chain reaction techniques to verify the identity of thescat and identified the diet using micro-histological analysis. Out of 83 scats, 36 were positively identified using molecu-lar analysis as originating from common leopards and 47 were leopard-negative. Wild prey contributed more than 82%of the common leopard diet, with the barking deer comprising most of this (33.5%). Domestic livestock was ranked sec-ond, at 17% of the diet. Other carnivores consumed small mammals (rodents, 27%) and livestock (19%), the proportionof which was higher than that consumed by common leopards in this region, although this difference was not significant.Our results suggest that small carnivores are no less responsible than common leopards for livestock depredation in thestudy area.

Konflikto su žmonėmis problema egzistuoja visame leopardų paplitimo areale. Tokių konfliktų valdymas Nepale yravienas iš valdžios veiklos prioritetų. Žmonių-leopardų konflikto masto nustatymui regione plėšrūnų ekskrementus rinkomežemutinėje saugomos Annapurna teritorijos dalyje (maždaug 400 km2 teritorijoje). Plėšrūno rūšis buvo identifikuotanaudojant polimerazinės grandininės reakcijos (angl. polymerase chain reaction, PCR) metodą. Plėšrūnų mitybiniaielementai buvo identifikuojami naudojant mikrohistologinius metodus. Molekulinės analizės būdu ištyrus 83 ekskrementųpavyzdžius nustatyta, kad 36 iš jų priklausė leopardams, 47 – kitiems gyvūnams. Daugiau kaip 82% leopardų racionosudarė laukiniai gyvūnai, iš kurių didžiausią dalį sudarė Kinijos muntjakai (33,5%). Naminiai gyvūnai sudarė 17%leopardų raciono. Kiti plėšrūnai mito smulkiaisiais žinduoliais, daugiausia graužikais (27%), ir naminiais gyvūnais (19%).Nustatyta, kad leopardų racione naminių gyvūnų dalis buvo šiek tiek mažesnė nei kitų plėšriųjų žinduolių racione. Mūsųrezultatai rodo, kad tirtoje teritorijoje smulkieji plėšrūnai darė ne mažesnę žalą naminiams gyvūnams nei leopardai.

Keywords: genetics; diet; livestock depredation; conflict; micro-histological analysis

Introduction

Human-carnivore conflict occurs almost invariably whenhuman settlements overlap with the ranges of largemammalian predators (McKenna and Bell 1997;Wozencraft 2005; Andheria 2006; Henschel 2008; Aryaland Kreigenhofer 2009; Eizirik et al. 2010; IUCN 2012;Koirala et al. 2012). Nepal provides prime habitat forlarge cat species such as snow leopards (Panthera uncia),common leopards (Panthera pardus), clouded leopards(Neofelis nebulosa), tigers (Panthera tigris tigris), andseveral species of small cats (Henschel 2008; Aryal andKreigenhofer 2009; IUCN 2012). Snow leopards arerestricted to higher elevations, and tigers to lower eleva-tions in Nepal (Oli 1994; Smith, Ahern, and

McDougal 1998). Common leopards, on the other hand,are the most common felid in Nepal, with a widespreaddistribution spanning both forest or heavy cover as wellas open country across a range of altitudes (up to 4400melevation) (Odden and Wegge 2005; Henschel 2008;Aryal and Kreigenhofer 2009; Koirala et al. 2012).

Common leopards enter into conflict with humansacross their range countries (Nowell and Jackson 1996;Mishra 1997; Aryal and Kreigenhofer 2009; Koirala et al.2012). In Nepal, human–carnivore conflict is an ongoingproblem, with many livestock being taken by predators(Henschel 2008; Aryal and Kreigenhofer 2009;Koirala et al. 2012; Aryal, Raubenheimer, Ji et al.2012). Consequently, the government of Nepal has

*Corresponding author. Email: r.k.koirala@massey.ac.nz

Zoology and EcologyVol. 22, Nos. 3–4, September–December 2012, 137–143

ISSN 2165-8005 print/ISSN 2165-8013 onlineCopyright � 2012 Nature Research Centrehttp://dx.doi.org/10.1080/21658005.2012.744864http://www.tandfonline.com

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formulated a compensation plan to recompense citizensfor livestock losses to carnivores, although the plan hasyet to be fully implemented (DNPWC 2009; Khatri2010). A key aspect of its implementation is thedifficulty of obtaining accurate information on livestocklosses. Often, the identity of the predator is unknown,and in other cases, local people withhold informationfrom data collectors or distort the exact figure of live-stock losses (Aryal, Sathyakumar, and Schwartz 2010;Koirala et al. 2012). In particular, there is a risk that thenumber of livestock killed is exaggerated to fraudulentlyincrease the level of compensation claimed from thegovernment (Bhatnagar, Wangchuk, and Jackson 1999;Namgail, Fox, and Bhatnagar 2007; Aryal andKreigenhofer 2009; Aryal, Sathyakumar, and Schwartz2010). There is therefore an urgent need for methods toindependently assess the scale and details of livestocklosses to carnivores.

In the southern belt of the Annapurna ConservationArea (ACA), Nepal, there are several predator species,including the common leopard, clouded leopard, jackal,yellow-throated Himalayan martin, common palm civet,and jungle cats (Giri et al. 2011; Koirala et al. 2012).Among these, the common leopard is believed to be themain predator of livestock and has therefore been thetarget of retaliatory killings (Koirala et al. 2012). In thisstudy, we aimed to obtain an independent assessment ofthe extent of livestock depredation in the lower belt ofthe ACA and the role of common leopard in livestocklosses. We collected predator scat over an area of400 km2, used species-specific polymerase chain reaction(PCR) combined with morphological analysis to establishthe identity of each scat, and subjected the samples tomicro-histological analysis for diet identification. Werelate our results, obtained using non-invasive scat(Foran, Crooks, and Minta 1997; Waits and Paetkau 2005)and diet identification, to the results of an earlier studythat used social interviews to gather information on thepatterns of livestock depredation by different carnivoresin the study region (Koirala et al. 2012).

Material and methods

Study area

The ACA is situated at 28°13′18″ to 29°19′48″ N latitudeand 83°28′48″ to 84°26′24″ E longitude. It is Nepal’s firstconservation area and largest protected area (Figure 1(a)and (b)), covering an area of 7629 km2. The ACA rangesfrom 1000 to 8091m in elevation, covering theTrans-Himalayan region to the subtropical region (Aryal,Raubenheimer, Sathyakumar et al. 2012). The area has ahigh biological diversity (Aryal 2009; Giri et al. 2011;Koirala et al. 2012) (Figure 1(a) and (b)). Amongresident species are the wolf (Canis lupus), musk deer(Moschus chrysogaster), common leopard (P. pardus),barking deer (Muntiacus muntjak), goral (Nemorhaedusgoral), Himalayan black bear (Selenarctos thibetanus),

lynx (Felix lynx), Jungle cat (Felis chaus), otter(Lutra spp.), yellow-throated marten (Martes flavigula),langur (Presbytis entellus), and Himalayan serow (Capri-cornis thar) (Aryal 2009; Giri et al. 2011; Koirala et al.2012; Aryal, Raubenheimer, Ji et al. 2012). The region isalso inhabited by socially and culturally diverse groups ofpeople (ACAP 2010), the majority of these people beingdependent on agriculture and animal husbandry forsubsistence (Koirala et al. 2012).

Data collection

Scat samples were collected across the southern belt of theACA from April 2009 to June 2010 (Figure 1(a)–(c)). Thesurvey covered an area of 400 km2. Scat samples werecollected along walking trail transects, each of which wassampled only once. A Geographical Position System loca-tion was recorded for each fecal sample (Figure 1(c)). Thesamples were air dried and put in 95% ethanol solution forgenetic analysis.

DNA extraction and species identification of scats

All collected scat samples were transported to thelaboratory of the Centre for Molecular Dynamics in Kath-mandu, Nepal (http://www.cmdn.org.np). Peripheral layersof scat samples were used for DNA extraction by QiagenQIAamp DNA Stool kit (Qiagen, Valencia, CA, USA).Scats were lysed in Lysis Buffer ASL and the supernatantwas reacted with the Inhibit EX tablet to adsorb PCRinhibitors of the scat. The supernatant was then reactedwith Buffer AL and Proteinase K and incubated for atleast 10min at 70 °C for further processing. The lysatecontaining free crude DNA was then retained on the silicamatrix of the spin column and eluted to obtain DNA.

A 130 bp leopard-specific target within NADH4region of mitochondrial DNA was PCR-amplified usinga forward primer (NADH4 F′) 5′-TRATAGCTGCYT-GATGAC-3′ and a reverse primer (NADH4 R′) 5′-GTTTGTGCCTATAAGGAC-3′ for species identification(Mondol et al. 2009).

PCR of 7 μl was prepared, containing 3.5 μl of 2XQiagen Multiplex PCR Master mix, 0.7 μl of 5XQiagen’s Q-Solution, 0.2 μl of each forward and reverseprimer to which 2.5 μl of extracted undiluted DNA wasadded. The PCR had the following thermo-cycling con-ditions: 95 °C for 15min followed by 50 cycles of 95 °Cfor 30 s, 52 °C for 30 s, and 72 °C for 45 s with the finalextension at 72 °C for 10min. The PCR products wereanalyzed in 2% agarose gel along with a known leopard-positive sample.

The genetic identification of scat by using fecal DNAallowed us to distinguish between leopard-positive samplesand leopard-negative samples, since no primers specific toother carnivore species were used. There is a possibility,however, that a portion of the leopard-negative resultsmight have arisen through a failed test (e.g. no viable DNAextracted). We therefore subjected all leopard-negativescats to a secondary, detailed morphological analysis. A

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subset of these was positively identified as belonging to car-nivores other than leopards, whereas a minority was toobadly fractured for reliable morphological analysis. For

purposes of dietary analysis, we assumed that the lattergroup belonged to carnivores other than the common leop-ard, based on the negative genetic analysis.

Figure 1. Location of the study area in Nepal (a), Annapurna Conservation Area and Kaski district (b), and collection sites ofleopard and other carnivore scats in relation to land use patterns in the study area.

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Diet analysis of common leopard and other carnivores

Following species identification, the scats were taken tothe Wildlife Laboratory at the Institute of Forestry,Pokhara, Nepal, for diet analysis. Standard micro-histological methods were used to identify prey throughthe scat hair samples by reference to hair samples fromknown prey species of leopards and other predators(Mukherjee, Goyal, and Chellam 1994; Aryal andKreigenhofer 2009; Bahuguna et al. 2010; Aryal,Hopkins et al. 2012), as well as reference photographs ofmammalian hair (Bahuguna et al. 2010). The scat sam-ples were first washed with hot water and thoroughlyair-dried, and then put in ether for one hour to removewax deposits and any remaining moisture. Finally, thehairs were treated in xylol for 24 h and culticular andmedullary structures were observed as in previousstudies (Mukherjee, Goyal, and Chellam 1994; Aryaland Kreigenhofer 2009; Aryal, Hopkins et al. 2012).Twenty hair samples were taken from each scat to com-pare with the prepared reference hairs and the referencephotographs. The residue composition of the feces interms of the frequency of occurrence of the prey speciesin the scat samples was extrapolated and calculated(Mukherjee, Goyal, and Chellam 1994; Aryal andKreigenhofer 2009; Aryal et al. 2012). While reliable interms of presence/absence measures, this technique issubject to error in estimating amounts of different preyeaten because large mammal prey and small prey itemsare underestimated and overestimated, respectively(Ackerman, Lindzey, and Hemker 1984). To compensatefor this, a correction factor for estimating original dietcomposition and proportional biomass consumption wasused. The correction factor (Y) was derived byAckerman, Lindzey, and Hemker (1984) and is describedby the equation (Y= 1.980 + 0.035X). The average unitweight of prey species (X) was taken from Karanth andSunquist (1995), Prater (1993), and Schaller (1977).Then, the relative biomass consumed by commonleopards and other carnivores was calculated. AStudent’s t-test was used to test for differences in thebiomass of different prey species consumed. We usedSPSS version 16.0.

During the field survey, we canvassed the opinion oflocal people on whether wildlife in the area hadincreased or decreased in recent years, using thequestionnaire technique described in Koirala et al.(2012). This survey was performed in 12 villages onrandomly selected respondents.

Results

Altogether, 83 scat samples were collected from the studyarea (Figure 1 (c)). Of these, 36 were genetically identi-fied as having originated from common leopards(Figure 2) while the remaining scats (n= 47) wereleopard-negative (Figure 2). Thirty of these wereconfirmed using detailed morphological analysis to

belong to species other than the common leopard,whereas 17, which were too fractured for morphologicalanalysis, were assumed to belong to species other thanthe common leopard. We made this assumption reasoningthat these scats had already been identified as leopard-negative using molecular techniques.

The pattern of distribution of different scats revealedthat leopards were distributed throughout the study area.The forested area near the villages was found to havethe highest leopard activity. The highest and lowest ele-vations of the scat distribution of leopards in the studyarea were 2513m in Bamboo (Ghandruk) and 1571m(Sickles), respectively.

Diet of common leopards

Natural prey contributed more than 82% of the commonleopard diets (Table 1). Estimation of the relative biomasscontribution of different prey species in the leopard dietshowed that the barking deer (33.5%), Himalayan serow(24%), and goral (20%) contributed the highest propor-tion, followed by domestic livestock (18%) (Table 1).The biomass of different prey species consumed by thecommon leopard was significantly non-random (t= 2.9,df = 7 p = 0.02), with barking deer being more commonthan other prey species (Table 1).

The diet of other carnivores

In our sample, the diet of carnivores other than of thecommon leopard consisted predominately of smallmammals and livestock. The forest mouse comprisedthe highest biomass contribution (27%), followed bygoral (21%), and barking deer (18%). Domestic animalscontributed 19% of the total biomass. The biomassconsumption of different prey species by other carni-vores in the region was significantly different (t= 3.89,df = 7, p= 0.006), with rodents and small domesticlivestock being more common than other prey species(Table 2).

The pattern of prey consumption by the commonleopard and other predators did not differ significantly(t= 0.1, df = 9, p= 0.92) (Tables 1 and 2). Other carni-vore species consumed a greater amount of domesticlivestock (28%) than common leopards did, but the dif-ference was not significant (t= 0.51, df = 4, p= 0.64).

Discussion

This is the first study of the common leopard diet in theACA region based on species-specific PCR. Our studyshowed that of the 83 samples we collected, all of whichmight have passed for the common leopard on casualinspection, 47 were suggested by PCR not to belong tocommon leopards and this conclusion was confirmedusing a detailed morphological analysis for 30 of these.This confirms the conclusion that scats can readily bemisidentified in the field when several species co-occur(Janecka et al. 2008; Karmacharya et al. 2011) andhighlights the need for caution.

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Our dietary analysis showed that the primaryprey of carnivores other than common leopards wassmall mammals such as rodents, whereas the mainprey for common leopards was the barking deer. Amajority of the local people interviewed (n = 157 outof 189) stated the perception that the prey popula-tion was declining because of habitat degradation,

disturbance, and poaching in some areas. The pres-ence of a variety of wild prey species, includinggoral and birds, in the carnivores’ diet suggests thatthe conservation and management of alternative preyspecies is vital for the conservation of the carni-vores and reduction of the human–common leopardconflict in the ACA.

Figure 2. Electrophoregrams of common leopard species-specific PCR products obtained amplifying a 130 bp common leopard-specific NADH4 region. L=DNA Ladder; PPR= known common leopard positive control; samples 28–56; NTC= no template control(negative control).

Table 1. Frequency of occurrence and relative biomass contributed by prey species identified in the scat of common leopard(n= 36).

Prey species Frequency of occurrence (%) CF (Y= 1980 + 0.035X) Relative biomass consumeda

Barking deer (Muntiacus muntjak) 35.56 2.68 33.5Himalayan serow (Capricornis thar) 13.33 5.17 24.2Goral (Naemorhedus goral) 17.78 3.21 20.0Sheep (Ovis spp.) 4.44 2.68 4.2Cow (Boss spp.) 6.67 4.78 9.6Goat (Capra spp.) 2.22 2.51 2.0Birds 6.67 2.02 4.7Dog (Canis spp.) 2.22 2.33 1.8Unidentified 11.11

aCorrected using the formula of Ackerman, Lindzey, and Hemker (1984).

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Our study also suggested that other carnivore speciesare responsible for killing a good deal of livestock in thearea, at least as much as the common leopard. This is sig-nificant, because, a recent survey established that localpeople in the region suspect the common leopard to be themain livestock predator (Koirala et al. 2012). Our objec-tive analysis using non-invasive techniques for positiveidentification of leopard scat has implications for manag-ing human–wildlife conflicts. Specifically, it suggests thatthere might be a degree of unwarranted antagonismtowards the common leopard among local people in thisregion, and education together with objective analysis offuture livestock losses might help to reduce the risk ofrevenge killings of the common leopard.

The development of objective molecular techniquesfor scat identification offers opportunities for understand-ing rapid alterations in the food web structure caused byanthropogenic impacts such as climate change and habi-tat degradation. It is interesting that our survey revealeda perception among local people that the populations ofmain prey species (e.g. barking deer) had been decliningin the area, suggesting major recent ecological distur-bances. This is a cause for concern, because a reductionin natural prey will increase the risk that scarce andendangered species are taken by predators. It will alsoincrease the likelihood of livestock predation, and eventhe perception that this might be the case could intensifyhuman–wildlife conflict in this ecologically sensitiveregion. Longitudinal studies of carnivore diets using themethods adopted in the present paper will provide asensitive and objective measure of such changes.

Acknowledgments

The project was funded by the Conservation Leaders MemorialCentre of Excellence (MemCoE) at the Institute of Forestry/Tribhuvan University, Nepal, in collaboration with VirginiaTech/Yale University and Principia College (funded by USAID/HED). Our sincere appreciation goes to Mercella Kelly(Virginia Tech, USA), A.L. (Tom) Hammett (Virginia Tech,USA), and Abadhesh Singh (Coordinator MemCoE) for theirsupport during the study period. We thank Centre forMolecular Dynamics, Nepal (CMDN), for genetic work toidentify carnivores and to ACA Project (ACAP)/National Trustfor Nature Conservation, Pokhara for research permission. Wethank IDEA WILD for providing field equipment, Rufford

Small Grant Foundation (UK), Massey University ResearchFund (New Zealand), and NUFU Project IOF for part of fieldsupport, and the Institute of Forestry, Pokhara (TribhuvanUniversity), Nepal, for lab work and other administrativesupport during the project period. Finally, we would like togratefully acknowledge, WWF, EFN (Education for NatureProgram) for professional development grant to participate inshort courses to enhance knowledge and skills in conservation.

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