In North America and Eurasia, the most ancestral populations of species often occur in the southern portions of their ranges (Hewitt, 2000;Petit et al., 2003). These southern regions tend to be the most climatically stable and therefore served as refugia during the heights of ice-age glaciations. In contrast, northern regions were associated with dramatic climate changes, fueling an alternation of range expansions and range contractions or extinctions in many species (Hewitt, 2000;Hofreiter & Stewart, 2009). Populations that today are widespread across northern latitudes often trace their origins to relatively recent, late Pleistocene expansion events from

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Background The Asiatic wild dog or dhole (Cuon alpinus) is a highly elusive, monophyletic, forest dwelling, social canid distributed across south and Southeast Asia. Severe pressures from habitat loss, prey depletion, disease, human persecution and interspecific competition resulted in global population decline in dholes. Despite a declining population trend, detailed information on population size, ecology, demography and genetics is lacking. Generating reliable information at landscape level for dholes is challenging due to their secretive behaviour and monomorphic physical features. Recent advances in non-invasive DNA-based tools can be used to monitor populations and individuals across large landscapes. In this paper, we describe standardization and validation of faecal DNA-based methods for individual identification of dholes. We tested this method on 249 field-collected dhole faeces from five protected areas of the central Indian landscape in the state of Maharashtra, India. Results We tested a total of 18 cross-species markers and developed a panel of 12 markers for unambiguous individual identification of dholes. This marker panel identified 101 unique individuals from faecal samples collected across our pilot field study area. These loci showed varied level of amplification success (57–88%), polymorphism (3–9 alleles), heterozygosity (0.23–0.63) and produced a cumulative misidentification rate or PID(unbiased) and PID(sibs) value of 4.7 × 10−10 and 1.5 × 10−4, respectively, indicating a high statistical power in individual discrimination from poor quality samples. Conclusion Our results demonstrated that the selected panel of 12 microsatellite loci can conclusively identify dholes from poor quality, non-invasive biological samples and help in exploring various population parameters. This genetic approach would be useful in dhole population estimation across its range and will help in assessing population trends and other genetic parameters for this elusive, social carnivore.

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24Faecal samples have become important non-invasive source of information in wildlife 25 biology and ecological research. Despite regular use of faeces, there is no universal protocol 26 available for faeces collection and storage to answer various questions in wildlife biology. 27 We collected 1408 faeces from ten different species using a dry sampling approach, and 28 achieved 94.87% and 86.02% success rate in mitochondrial and nuclear marker 29 amplifications. We also suggest a universal framework to use the same samples for different 30 use. This protocol provides an easy, quick and cheap option to collect non-invasive samples 31 from species living at different environmental conditions to answer multidisciplinary 32 questions in wildlife biology. 33 34 35 Keywords: Non-invasive wildlife research, species biology, dry sampling, variable habitat, 36 field logistics. 37 38 39 40 41 42 43 44 45 46 Non-invasive samples, in particular faeces have become a regular choice in wildlife biology, 47 population monitoring and ecological research globally. Advantages of faecal sample-based 48 wildlife research include easy sample collection, access to large sample size and spatio-49 temporal coverage. Historically, large scale use of faeces in wildlife biology started with 50 dietary analysis of animals 1 but the introduction of advanced molecular tools added a new 51 dimension to non-invasive research. These molecular tools have allowed biologists to 52 investigate questions regarding population genetics 2,3 , species distribution 4 , demography 5,6 , 53 evolutionary biology 7 and wildlife forensics 8 . In more recent time, faecal samples have been 54 used in addressing various questions related to wildlife physiology including endocrinology 55 and reproductive capacity 9,10 , along with parasitology 11,12 , disease dynamics 13 and 56 conservation genomics 14 . The sampling and storage demands of various questions in non-57 invasive wildlife research have led to a gradual development of faecal sampling and storage 58 protocols. A number of logistical factors including collector's safety, storage in the field, 59 shipping samples from remote field areas with different environmental conditions etc. have 60 been considered while gradual development of these protocols. 61Over the years, a number of faeces collection and storage approaches has been used in 62 wildlife research that are broadly classified into three categories: a) dry sampling (for 63 example simple drying 15 , silica preservation 16 ); b) wet sampling (ethanol collection 17 ; TNE 64 and DMSO buffer 18 ; DETs solution 19 ; RNA later 20 ) and c) two-step approach 21 (see Table 65 1 for details). While all of these approaches have been used in wildlife research, they have 66 several logistical limitations making their implementation in the field challenging. For 67 example, sampling with silica beads has advantages in post-collection sample transport and 68 storage 22 but is not cost effective as it requires large amount of silica beads to keep samples 6...