The Role of Genetics

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O U T L I N E 27.1 Conservation Genetics of Snow Leopards

Snow Leopards http://dx.doi.org/10.1016/B978-0-12-802213-9.00027-4

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27.2 Diet Reconstruction of Snow Leopard Using Genetic Techniques 375

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Copyright © 2016 Elsevier Inc. All rights reserved.

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Subchapter27.1

Conservation Genetics of Snow Leopards Anthony Caragiulo*, George Amato*, Byron Weckworth** *American Museum of Natural History, Sackler Institute for Comparative Genomics, New York, NY, USA **Snow Leopard Program, Panthera, New York, NY, USA

INTRODUCTION The goal of conservation biology is to reduce the current rates of extinction and preserve biodiversity. The primary threat to many endangered species is small population size. At small population sizes, stochastic effects related to demographic, environmental, and genetic consequences increase extinction risks. Genetic diversity, along with species and ecosystem diversity, is recognized by the IUCN as one of the top three forms of biodiversity requiring conservation (Mcneely et al., 1990). Conservation geneticists apply genetic theory and techniques to preserve endangered species as dynamic entities, capable of coping with environmental change and thus minimizing their risk of extinction (Frankel and Soulé, 1981). The application of molecular tools to conservation research provides biologists, managers, and policy makers with insights into the drivers of extinction, which helps to inform appropriate management practices (Sarre and Georges, 2009). Snow leopards (Panthera uncia) are an umbrella species of the high elevation regions of Central Asia, and a keystone for maintaining biodiversity within these fragile ecosystems

amid the impacts of climate change and human perturbation (Li et al., 2014). Snow leopards are currently categorized as “Endangered” by the International Union for the Conservation of Nature (IUCN) and the Convention on International Trade in Endangered Species (CITES) Appendix I. This categorization is a result of declining snow leopard numbers and loss of habitat stemming from prey loss, conflict with humans, and poaching for hides and use of parts in traditional medicine. Snow leopards are believed to have been extirpated from as much as 15% of their historic range, and in some areas their numbers have declined by as much as 20% in the late twentieth century (Mccarthy and Chapron, 2003). Snow leopards are generally solitary and maintain stable home ranges delineated using markings such as scat, urine, and scrapes. A clear understanding of patterns of snow leopard population trends and genetic diversity is critical for guiding conservation initiatives that will ensure their long-term persistence. In this chapter we briefly review the most widely used genetic tools available for snow leopards, summarize the most important published studies of snow leopard genetics, and outline priority research questions and needs that would fill



Major genetic tools available for snow leopard conservation

important gaps in our use and understanding of conservation genetics to support snow leopard conservation.

MAJOR GENETIC TOOLS AVAILABLE FOR SNOW LEOPARD CONSERVATION Snow leopards are a cryptic species and naturally occur at low densities, making them difficult to study. These characteristics make it especially difficult to collect blood and tissue samples for genetic studies. Advances in molecular biology and genetics have made DNA collection much easier, as noninvasive molecular techniques have increasingly become the norm in studying large carnivores (Taberlet et al., 1996; Waits and Paetkau, 2005). Noninvasive is an umbrella term and refers to samples obtained without direct observation or handling of the target animal, with the most common sample types being hair and scat. The benefit of noninvasive sampling is that the target species never has to be directly observed or handled, making it ideally suited for studying snow leopards. Noninvasive sampling also reduces the risks associated with immobilizing and handling rare and endangered species. The major detriment, however, is the poor DNA quality obtained from noninvasive samples. Advanced molecular techniques provide workarounds for obtaining reliable species and individual identifications from these samples, but extra care needs to be taken when analyzing data from noninvasive samples (Broquet et al., 2007; Miquel et al., 2006; Ruell and Crooks, 2007; Taberlet et al., 1999; Waits and Paetkau, 2005). Hair samples can be collected through hair corrals, rub stations, and hair snares, and have been used to monitor a variety of carnivores including ursids, felids, canids, and mustelids (Kendall and Mckelvey, 2008). Scat has also been an effective means to monitor large carnivores,

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due to its ubiquity in nature and source of target DNA (Foran et al., 1997a). Additionally, scat provides the ability for dietary analysis, both through traditional methods of prey identification through undigested material (i.e., hair, bones) and molecular methods (Emmons, 1987; Farrell et al., 2000; see Chapter 27.2). Given their prevalence for marking predictable sites on the landscape with scat, and the cool, dry conditions that typify their habitat, noninvasive sampling for snow leopards is comparatively easy versus other large carnivores and may yield higher quality DNA for downstream analyses. Hair and scat are a source of mitochondrial and nuclear DNA and can be used for both species and individual identification (Farrell et al., 2000; Foran et al., 1997b; Reed et al., 1997). Hair samples provide DNA in dried epithelial cells (dander) that cling to the shaft, and a higher proportion of DNA from cells within the hair follicle. Scat samples provide DNA from the epithelial cells lining the large intestine of the target species. The epithelial cells are sloughed off as feces move through the animal’s colon and comprise the outer covering of the resultant scat. Urine has also been utilized for noninvasive monitoring of populations, as DNA may be obtained from epithelial cells lining the urinary tract shed during urination, however, this method is less popular than hair and scat due to limitations in collection. Namely, urine has mostly been used for species inhabiting snowy areas and collected from urine-covered snow (Hedmark et al., 2004; Valiere and Taberlet, 2000; Van Der Hel et al., 2002), thereby limiting its ubiquity compared to hair and scat. However, when able to be collected, urine has been shown to provide quality genetic information about species and populations (Hedmark et al., 2004; Valiere and Taberlet, 2000). Additionally, scent spray marking, particularly in felids such as snow leopards, can also provide the raw genetic material from the field for identifying species and individuals (Caragiulo et al., 2015).

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Noninvasive samples are an excellent source of mitochondrial DNA (mtDNA), which can be used for reliable species identification from numerous vertebrate species (Cronin et al., 1991; Farrell et al., 2000; Foran et al., 1997a; Kitano et al., 2007; Melton and Holland, 2007; Mukherjee et al., 2007; Paxinos et al., 1997). Mitochondria are numerous within a typical cell and contain small circular haploid DNA molecules that are maternally inherited and usually transmitted without recombination (Barr et al., 2005). Additionally, mtDNA evolves more rapidly than most nuclear DNA, resulting in the accumulation of differences between closely related species (Brown et al., 1979; Hebert et al., 2004). The abundance, ease of purification and sequencing, and interspecific sequence conservation make mtDNA ideal for identifying species from noninvasive samples (Foran et al., 1997a; Chaves et al., 2012). Mitochondrial DNA, specifically the cytochrome oxidase I gene region, provides DNA barcodes for the identification of samples from unknown species (Eaton et al., 2010; Hebert et al., 2003), and interspecific variation can be resolved from short DNA sequences, which is an important aspect when dealing with highly degraded DNA from noninvasive samples. Nuclear DNA is less commonly used for species identification and is less efficient for species identification than mtDNA markers (Rastogi et al., 2007); however, some nuclear markers (e.g., internal transcribed spacer regions) have shown promise for species identification (Schoch et al., 2012). Purification of DNA from noninvasive samples has advanced over the years, but the fact remains that it is still highly degraded and fragmented due to its source and the environmental conditions to which it is subjected prior to collection. Mitochondrial DNA holds utility for discriminating between species, but analysis of nuclear DNA is necessary for identifying individuals and delineating populations. The most commonly used molecular marker is the microsatellite: short, tandemly repeated (usually between one and five base pair repeats) nuclear sequences

that operate with traditional Mendelian inheritance (Jarne and Lagoda, 1996). They are selectively neutral and noncoding, making them ideal for examining population structure and gene flow (Rannala and Mountain, 1997), and many loci have been developed for snow leopards (e.g., Janecˇ ka et al., 2008; Waits et al., 2007). Advances in sequencing technology have opened the door for single nucleotide polymorphisms (SNPs) to also be used similarly to microsatellites. SNPs are the most common type of genetic variability in most genomes and offer the potential for genome-wide scans of selectively neutral or adaptive variation with simple mutation models, powerful analytical methods, and application to noninvasive and historic DNA (Morin and Mccarthy, 2007; Morin et al., 2004; Morin et al., 2009). SNPs hold an advantage over microsatellites in that they are less prone to amplification error due to the single nucleotide nature versus longer sequences of tandem repeats in microsatellites, SNP error can be more easily quantified, and there is no user bias in the scoring of SNP genotypes. The disadvantage of SNPs is that panels of informative SNP loci have not been developed for many vertebrate species and this may need to be done de novo for a target species, whereas microsatellite panels exist for many vertebrate taxa.

MOLECULAR MARKERS FOR DETERMINING POPULATION STRUCTURE, CONNECTIVITY, AND PATTERNS OF GENE FLOW All the previously mentioned molecular markers can be used to assess population structure, connectivity, and patterns of gene flow from noninvasively collected samples, and each addresses questions at different temporal scales. For example, mtDNA can be used to examine matrilineal lineages and pedigrees within species. Additionally, mtDNA is most commonly used in phylogeographic studies to examine the

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Conservation genetics and molecular ecology in snow leopards to date

spatial distribution and divergence of populations (Avise et al., 1987; Avise, 1992). Haplotypic variation among populations and the pattern of mutational accumulation often provide insight into the evolutionary history of a species, as they have for numerous wild felid species such as pumas (Caragiulo et al., 2014a; Culver et al., 2000), cheetahs (Charruau et al., 2011), tigers (Driscoll et al., 2009; Luo et al., 2004; Luo et al., 2010), jaguars (Eizirik et al., 2001), leopards (Uphyrkina et al., 2001), clouded leopards (Kitchener et al., 2006), and lions (Barnett et al., 2006), but not yet on snow leopards. These analyses examine shared haplotypes amongst populations and stepwise mutational processes to look at the progression and divergence of the species on a spatial and geographic scale. The major drawback to all of these molecular markers is that they are not interchangeable in their application. In order for meaningful comparisons between studies and species, the same markers need to be used. For instance, two studies on snow leopards must use the same microsatellite loci, or mtDNA markers, or SNP panel for comparison or combination of datasets for large-scale applications. Snow leopard conservation requires a coordinated effort regarding all genetic work to allow comparison between studies that use the same genetic markers to link local populations to amass as close to a rangewide dataset as possible.

GENETIC ANALYSIS OF NONNEUTRAL (ADAPTIVE) GENETIC VARIATION AND IMPLICATIONS FOR ADAPTATION TO A CHANGING ENVIRONMENT The aforementioned measures of gene flow and connectivity are all based on selectively neutral molecular markers. These methods provide information about microevolutionary factors such as gene flow and can prioritize conservation units, but do not inform the potential

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for adaptation to changing environments. For this, one of the most common markers used is the major histocompatibility complex (MHC), which is a large cluster of loci involved in recognizing pathogen antigen molecules and regulating immune response (Frankham et al., 2010). Selection at the MHC favors heterozygotes to maintain high levels of genetic diversity; however, diversity is commonly lost by drift in small populations and this leads to a decrease in the ability to combat disease and potential extirpation (Frankham et al., 2010). Examining the MHC for select populations can indicate their ability to combat introduced or emergent pathogens For example, low diversity at the MHC is commonly cited as the cause of Tasmanian devil (Sarcophilus harrisii) decline in Australia due to a transmissible clonal facial tumor (Siddle et al., 2010). Genetic diversity in the MHC may be particularly critical for snow leopards as climate change hastens the emergence and spread of new diseases (Altizer et al., 2003), most imminently at the high altitudes they inhabit.

CONSERVATION GENETICS AND MOLECULAR ECOLOGY IN SNOW LEOPARDS TO DATE Despite its high profile of a charismatic carnivore, snow leopard information is scarce and difficult to obtain due to its cryptic nature and remote habitat. Additionally, snow leopard range largely encompasses areas of political turmoil, yielding additional layers of complexity in their study, not the least of which includes difficulties for genetic work requiring transporting biological material for study. A literature survey on snow leopards yields surprisingly few studies for an animal with a such a large ecological footprint; nonetheless, we attempt to characterize and summarize what is known regarding snow leopards from a genetic standpoint, and address the importance of this work as a foundation for future snow leopard research.

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One of the first studies to characterize snow leopard genetics was Zhang et al. (2007), which developed snow leopard-specific primers to amplify a section of the control region in the mitochondrial genome. The control region is highly variable, and species-specific primers are often necessary to characterize intraspecific variation. While Zhang et al. (2007) used skin and hair samples from 12 snow leopards from China, the primers developed are also useful in wildlife forensics, species identification, and population genetic studies using noninvasive snow leopard samples. They identified six haplotypes and six parsimony-informative sites within their 411 base pair (bp) sequence, making this primer set a good candidate for examining mitochondrial diversity and phylogeographic patterns of snow leopards in other portions of their range. Wei et al. (2009) went a step further and amplified the entire mitochondrial genome of the snow leopard, allowing researchers to design primers to examine any mitochondrial gene region for phylogeography and evolutionary studies of the Pantherines. As expected, the snow leopard mitochondrial genome structure is similar to other felids. Wei et al. (2009) performed a phylogenetic analysis using 10 felid species and approximately 4000 bp of mitochondrial DNA spanning seven genes and resolved the snow leopard as sister to lions (Panthera leo). In contrast, results from Caragiulo et al. (2014b), which used entire mitochondrial genomes for every Pantherine, identified snow leopards as sister to the monophyletic clade of leopards (Panthera pardus) and lions. A study by Davis et al. (2010), used a supermatrix of nuclear and mitochondrial genes with sex chromosome sequences, and contradicted both of these relationships, firmly placing snow leopards as sister species to tigers (Panthera tigris). The Davis et al. (2010) study compared the most comprehensive amount of genetic data, and the relationship between snow leopards and tigers was corroborated by an analysis using both genetic and morphological data by Tseng et al. (2013). The study of snow leopard evolutionary

history was enhanced through the development of mitochondrial primers, but has yet to be used to examine the demographic history of snow leopards, a vital aid toward their conservation and management. Whereas mtDNA provides information at the population and species level, finer-scale genetic analysis through individual identification is an essential advancement for studying snow leopards through noninvasive genetics. Individual identification of snow leopards was made possible through the development of a panel of polymorphic microsatellite loci, originally developed for domestic cats (Felis catus) (Waits et al., 2007). They reported 48 polymorphic microsatellite loci in snow leopards with 2–11 alleles per locus. Additionally, they identified 10 loci with significant power to discriminate among individuals. Identification of individuals through multiple microsatellite loci is used with mark-recapture statistics to estimate population numbers or the number of individuals in a given study area. Minimum numbers of individuals and population estimates of snow leopards through noninvasive genetic methods is incredibly useful given the elusiveness of snow leopards and the difficulty of direct observations in the wild, thereby potentially increasing the number of individuals that can be sampled. The reporting of polymorphic microsatellite loci was a first step in snow leopard conservation genetics, and it allowed for the identification of individuals from noninvasively collected samples. This work was advanced by further refinement of primers for seven of the loci identified in Waits et al. (2007) to be snow leopard– specific (Janecˇka et al., 2008). The snow leopard-specific microsatellite primers had a higher success rate when used with scat samples because they amplified shorter DNA regions and primed to a more specific sequence, rather than to the closely related sequence of the domestic cat. The study by Janecˇka et al. (2008) was one of the first to examine wild snow leopards

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Conservation genetics and molecular ecology in snow leopards to date

in different portions of their range (northwest India, central China, and southern Mongolia) using noninvasive genetic techniques. They surveyed a total transect length of 16.2 km and genetically identified 85 scat samples across those three geographic areas. Of the 85 genetically identified scat samples, 31 were snow leopards and equated to 10 individuals (2–4 males and 2 females in southwestern India; 1 of unknown gender in central China; and 3–5 males and 2 females in southern Mongolia); however, the individual from China yielded poor quality DNA and was not included in further analysis. Overall, they found low genetic diversity among the nine analyzed individuals, with 2.5–3 mean alleles per locus. Analysis of four mtDNA regions showed little genetic variability, with two haplotypes exhibited in the control region. All individuals sampled in southwestern India exhibited the two haplotypes, while central China and southern Mongolia samples exhibited the same singular haplotype. Due to small sample sizes, the study was merely a descriptive one, but provided the first baseline genetic information on the species. Janecˇ ka et al. (2008) was followed by the only other noninvasive genetics study on snow leopards (Karmacharya et al., 2011), which focused on Shey Phoksundo National Park (SPNP) and Kanchenjunga Conservation Area (KCA) in Nepal. A total of 71 scat samples were collected, with 19 of them genetically identified as snow leopard using mitochondrial DNA genes. From these, genotypes were generated using six microsatellite loci described in Janecˇ ka et al. (2008) for 10 samples, revealing nine individuals (three males, six females). The Karmacharya et al. (2011) study was also purely descriptive and provided baseline genetic information on snow leopards in the area. The authors suggested that their data be used to design a more in-depth population survey to estimate snow leopard abundance at the study sites. Advances in next-generation sequencing technology have increased the scope of genetic

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questions. Recently, 109 gigabases of total snow leopard sequence data with 40x coverage was compared to the genomes of the tiger and lion (Cho et al., 2013). This comparison highlighted a unique amino acid change in snow leopards that is consistent with adaptation to high altitudes. Specifically, snow leopards exhibited a specific genetic determinant in the EGLN1 gene (Met39  > Lys39), which is a human homolog for mediating high altitude adaptation. Amino acid changes in the EGLN1 gene, as well as the EPAS1 gene, account for hypoxia tolerance in naked mole rats and led Cho et al. (2013) to hypothesize the amino acid change observed in snow leopards confers an adaptive advantage to high altitude. Next-generation sequencing gives scientists the ability to look deeper into the genomes of organisms and understand complex relationships between genes and potential adaptations. Additionally, next-generation sequencing amplifies short stretches of DNA, which makes noninvasive samples well suited for this methodology because their DNA is already fragmented by its very nature. A few studies have compared the use of noninvasive genetic techniques to more traditional carnivore survey methods. McCarthy et al. (2008) compared in Kyrgyzstan and China the accuracy of Snow Leopard Information Management System (SLIMS) sign surveys to other abundance estimators, one of which was genetic analysis of snow leopard scat. The study found that noninvasive genetic methods of snow leopard abundance did not correspond to SLIMS sign surveys, with more snow leopards generally detected through genetic analysis. The authors concluded that none of the abundance estimators agree and estimating snow leopard numbers with confidence requires greater effort and better documentation. These findings are similar to those in Janecˇ ka et al. (2011) in which they estimated 5–6 individuals/100 km2 based on noninvasive genetics compared to 1.5 individuals/100 km2 based on camera trapping in the Gobi Desert of Mongolia.

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As demonstrated already, a major void remains in our understanding of snow leopard genetics. The published molecular work on snow leopards has almost entirely focused on the phylogenetic relationship of snow leopards among other cat species (e.g., Davis et al., 2010) or survey techniques in species and individual identification for diet and occupancy analyses (e.g., Janecˇ ka et al., 2008; Janecˇ ka et al., 2011; Mccarthy et al., 2008). The Janecˇ ka et al. (2008) and Karmacharya et al. (2011) studies exemplify the information gap with regard to snow leopard genetic studies. They are, to our knowledge, the only two population genetic studies on snow leopards, and the small sample sizes and distance between all sampling locales make them

inappropriate to build a comprehensive genetic network. The latter point speaks to the naturally low densities of snow leopards and the extreme difficulty in surveying their mountainous habitat. The solution to this problem requires more extensive sampling across a greater number of areas. To date, no published studies describe snow leopard phylogeography or population and landscape genetics, and the number of published snow leopard-specific genetic studies remains far below that of the other imperiled big cats. There is clearly an urgent need to initiate conservation genetic research to begin filling the gaps in our understanding of the molecular ecology of endangered snow leopards.

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Introduction

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Subchapter27.2

Diet Reconstruction of Snow Leopard Using Genetic Techniques Wasim Shehzad Institute of Biochemistry & Biotechnology, University of Veterinary & Animal Sciences, Lahore, Pakistan

INTRODUCTION Predation is a central interspecific relationship that can be studied by multidisciplinary approaches involving ecological, evolutionary, and behavioral sciences, leading to an understanding ecosystem function. Studies of carnivore diets can help evaluate resources used within an ecosystem (Mills, 1992) by characterizing prey selection with regards to prey availability, which in turn can provide an indicator of ecosystem stability. Accurate diet information can also be useful in understanding human-wildlife conflicts, such as those between large carnivores and human populations reliant on livestock (e.g., Bagchi and Mishra, 2006; Inskip and Zimmermann, 2009). Livestock depredation is a challenge throughout the range of the snow leopard (see Chapter 5), which results in hostility toward the animal from local communities (Mishra, 1997; Mishra et al., 2006; Inskip and Zimmermann, 2009) and retribution killings of snow leopards (Hussain, 2003; Bagchi and Mishra, 2006). To date, the diet of the snow leopard has been analyzed using a variety of classical methods. Inference from field surveys, questionnaires, and

interviews with local people can give an assessment of snow leopard predation and has been effectively used in some studies (e.g., Mishra, 1997; Namgail et al., 2007). But such studies may represent opinions and lack scientific rigor. Radio telemetry allows the study of snow leopard movements, home range, pattern of habitat utilization, and social organization (McCarthy, 2000), although locating the remains of killed prey in high, steep terrain is extremely difficult (Jackson, 1996). Johansson et al. (2015) assessed spatiotemporal variation in predation patterns of snow leopards and their kill rates in a GPS collaring study of snow leopards in Mongolia. However, that study is unique in its success and is not easily replicated across the snow leopard’s broad range. Examining feces may then represent the most readily available and easily collected source of diet information (Putman, 1984); this technique has been used extensively to study snow leopard diets (Oli, 1994; Lovari et al., 2009; Anwar et al., 2011). Such diet analysis requires the identification of undigested remains, bones, teeth, or hair in feces. There are two potential problems with fecal examination to assess snow leopard diets. The first relates

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to the accurate identification of snow leopard feces in the field, while the second deals with the limitations of accurately identifying the prey taxa.

possible prey items can prohibit accurate identification of remains.

LIMITATIONS OF CLASSICAL METHODS

Genetic methods may address both the problem of identifying the species that deposited the material and the prey remains they contain. Feces collected in the field are now easily validated by genetic analysis (Davison et al., 2002). Yet in several studies (e.g., Lovari et al., 2009; Anwar et al., 2011; Wang et al., 2014), snow leopard feces have been genetically validated and then prey content determined using conventional microscopic analysis, hence accurate and complete prey species identification remained a potential problem. DNA-based approaches are particularly suitable to overcoming this problem. Using feces as a source of DNA from food (Fernandes et al., 2008; King et al., 2008; Corse et al., 2010) provides a method for studying the feeding ecology of elusive and secretive animals such the snow leopard. The method uses universal primers, which requires no a priori knowledge of the diet to amplify prey DNA. The approach has been successfully implemented for herbivore (Valentini et al., 2009), yet the analysis of carnivore diets presents challenges, as predator DNA can be simultaneously amplified with prey DNA (Deagle et al., 2007). Furthermore, prey fragments might be rare in the fecal DNA extract, and consequently they have the tendency of being missed during the early stages of PCR, resulting in a PCR product containing almost exclusively the dominant sequence of predators (Green and Minz, 2005; Jarman et al., 2006). So far, two PCR-based approaches exist that could be used to study the snow leopard diet. In the first approach, the PCR amplification primers are designed for a particular prey species and successful PCR amplification product from

Snow leopard feces are identified in the field mostly on the basis of size, shape, location, and associated sign such as pugmarks, scrapes, or the remains of prey species near the feces (Bagchi and Mishra, 2006). Yet carnivore feces are quite similar in their morphological characteristics, and it is often difficult to differentiate the feces of sympatric carnivores (Hansen and Jacobsen, 1999; Spiering et al., 2009). Diet assessment based on such erroneous fecal identification may have far-reaching consequences in terms of conservation planning for snow leopards. Some studies (e.g., Long et al., 2007; Vynne et al., 2011) have used detection dogs trained to locate the feces of specific carnivores in the field. In another study, scat detection dogs were trained to distinguish snow leopard feces from other nontarget feces ex situ (Snow Leopard Trust, unpublished data), thus eliminating the cost and complications of bringing a dog to the field. Neither method is now in common use for snow leopards and may not be cost effective. Even in accurately identified feces, analysis of contents has many hurdles. Large bones and teeth are generally fragmented and therefore difficult to identify (Oli, 1993) from feces. Hair is commonly identified by comparisons with mounted reference specimens. However, this method is laborious and time consuming. Hairs from the same animal may also vary in structure according to their location on the body. Similarly, hair from several related species may possess similar characteristics (Oli, 1993). Finally, a reference collection that lacks specimens of all

GENETIC METHODS

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REFERENCES

a sample indicates the presence of DNA from the target; such an approach is referred as the targeted approach. To our knowledge, the PCRbased specific approach has not been used to study snow leopard diets. The second approach employs primers that bind to DNA regions conserved in a broad range of prey items and the PCR products amplified with these conserved primers are subsequently characterized; this can be thought of as the exploratory approach (see Shehzad et al., 2012a, b). The method uses a combination of a general primer for vertebrates (12SV5) that targets the 12rRNA gene of mitochondria for prey identification, and a blocking oligonucleotide (UnciB) sequence targeting the same gene and is highly specific to snow leopard DNA with a carbon 3 (C3) spacer at 3’end. The method has the potential to amplify all prey items present in the snow leopard diet, while addition of the blocking oligonucleotide limits the PCR amplification of snow leopard DNA present in a mixture of fecal DNA extract. To observe the rarest prey items in the diet the concentration of snow leopard blocking oligonucleotide may need to be as much as 20x the concentration of general primers for vertebrates. Such an approach requires a next-generation sequencing platform, because there is the potential to amplify hundreds of thousands of sequences of both predator and prey as required to elaborate all pray taxa present in the feces (Shehzad et al., 2012a). Investment in or use of such equipment can be costly, but the methodology can produce a far more detailed and accurate assessment of the snow leopard diet than previously possible.

CONCLUSIONS An unambiguous understanding of an endangered snow leopard’s diet is crucial for conservation planning for this species. To date, despite

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numerous studies, the diet of the snow leopard has been inadequately assessed due to misidentifying feces as being those of snow leopards, and the inherent inaccuracies of classic macro- or microhistological examination of fecal content. The methods described by Shehzad et al. (2012a) can address both these shortcomings through genetic techniques. A better understanding of the diet of the snow leopard will allow a more accurate assessment of the level of conflict between the cats and pastoralists who rightly or wrongly attribute their livestock depredation losses to snow leopards. Mitigating measures can then be designed that address a real, as opposed to a perceived, conflict. The techniques can also be employed to help assess opportunities to increase snow leopard numbers in areas where they have been reduced. Knowledge of diet composition and prey availability in such instances would help conservationists determine whether adequate wild prey is available to support the hoped-for increase in snow leopard populations. This would help avoid situations where increasing leopard numbers only result in escalating conflicts with livestock and humans, dooming the effort to failure. Conversely, where conflict is already high and conservation efforts focus on reducing livestock depredation (predator-proof corrals, better guard dogs, etc.), an accurate assessment of diet composition and wild prey availability would help avert unintended stress to snow leopards already facing inadequate food supplies to sustain their existing numbers.

References Altizer, S., Harvell, D., Friedle, E., 2003. Rapid evolutionary dynamics and disease threats to biodiversity. Trends Ecol. Evol. 18, 589–596. Anwar, M., Jackson, R., Nadeem, M., Janecˇka, J., Hussain, S., Beg, M., Muhammad, G., Qayyum, M., 2011. Food habits of the snow leopard Panthera uncia (Schreber, 1775) in Baltistan, Northern Pakistan. Eur. J. Wildl. Res. 57, 1077–1083.

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