Lion Localizer: A software tool for inferring the provenance of lions (Panthera leo) using mitochondrial DNA.

The illegal poaching of lions for their body parts poses a severe threat to lion populations across Africa. Poaching accounts for 35% of all human-caused lion deaths, with 51% attributed to retaliatory killings following livestock predation. In nearly half of the retaliatory killings, lion body parts are removed, suggesting that high demand for lion body parts may fuel killings attributed to human-lion conflict. Trafficked items are often confiscated in transit or destination countries far from their country of origin. DNA from lion parts may in some cases be the only available means for examining their geographic origins. In this paper, we present the Lion Localizer, a full-stack software tool that houses a comprehensive database of lion mitochondrial DNA (mtDNA) sequences sourced from previously published studies. The database covers 146 localities from across the African continent and India, providing information on the potential provenance of seized lion body parts. Lion mtDNA sequences of 350 or 1,140 bp corresponding to the cytochrome b region can be generated from lion products and queried against the Lion Localizer database. Using the query sequence, the Lion Localizer generates a listing of exact or partial matches, which are displayed on an interactive map of Africa. This allows for the rapid identification of potential regions and localities where lions have been or are presently being targeted by poachers. By examining the potential provenance of lion samples, the Lion Localizer serves as a valuable resource in the fight against lion poaching. The software is available at https://lionlocalizer.org.

Genomic analysis supports Cape Lion population connectivity prior to colonial eradication and extinction.

Cape lions (Panthera leo melanochaitus) formerly ranged throughout the grassland plains of the "Cape Flats" in what is today known as the Western Cape Province, South Africa. Cape lions were likely eradicated because of overhunting and habitat loss after European colonization. European naturalists originally described Cape lions as "black-maned lions" and claimed that they were phenotypically distinct. However, other depictions and historical descriptions of lions from the Cape report mixed or light coloration and without black or extensively developed manes. These findings suggest that, rather than forming a distinct population, Cape lions may have had phenotypic and genotypic variation similar to other African lions. Here we investigate Cape lion genome characteristics, population dynamics, and genetic distinctiveness prior to their extinction. We generated genomic data from 2 historic Cape lions to compare to 118 existing high-coverage mitogenomes, and low-coverage nuclear genomes of 53 lions from 13 African countries. We show that, before their eradication, lions from the Cape Flats had diverse mitogenomes and nuclear genomes that clustered with lions from both southern and eastern Africa. Cape lions had high genome-wide heterozygosity and low inbreeding coefficients, indicating that populations in the Cape Flats went extinct so rapidly that genomic effects associated with long-term small population size and isolation were not detectable. Our findings do not support the characterization of Cape lions as phylogeographically distinct, as originally put forth by some European naturalists, and illustrates how alternative knowledge systems, for example, Indigenous perspectives, could potentially further inform interpretations of species histories.

From Caves to the Savannah, the Mitogenome History of Modern Lions (Panthera leo) and Their Ancestors.

Lions (Panthera leo) play a crucial ecological role in shaping and maintaining fragile ecosystems within Africa. Conservation efforts should focus on genetic variability within wild populations when considering reintroduction attempts. We studied two groups of lions from two conservation sites located in Zambia and Zimbabwe to determine their genetic make-up, information that is usually unknown to the sites. In this study, we analysed 17 specimens for cytb and seven microsatellite markers to ascertain family relationships and genetic diversity previously obtained by observational studies. We then produced a standardised haplogroup phylogeny using all available entire mitogenomes, as well as calculating a revised molecular clock. The modern lion lineage diverged ~151 kya and was divided into two subspecies, both containing three distinct haplogroups. We confirm that Panthera leo persica is not a subspecies, but rather a haplogroup of the northern P.l. leo that exited Africa at least ~31 kya. The progenitor to all lions existed ~1.2 Mya, possibly in SE Africa, and later exited Africa and split into the two cave lion lineages ~175 kya. Species demography is correlated to major climactic events. We now have a detailed phylogeny of lion evolution and an idea of their conservation status given the threat of climate change.

The evolutionary history of extinct and living lions.

Lions are one of the world's most iconic megafauna, yet little is known about their temporal and spatial demographic history and population differentiation. We analyzed a genomic dataset of 20 specimens: two ca. 30,000-y-old cave lions (Panthera leo spelaea), 12 historic lions (Panthera leo leo/Panthera leo melanochaita) that lived between the 15th and 20th centuries outside the current geographic distribution of lions, and 6 present-day lions from Africa and India. We found that cave and modern lions shared an ancestor ca. 500,000 y ago and that the 2 lineages likely did not hybridize following their divergence. Within modern lions, we found 2 main lineages that diverged ca. 70,000 y ago, with clear evidence of subsequent gene flow. Our data also reveal a nearly complete absence of genetic diversity within Indian lions, probably due to well-documented extremely low effective population sizes in the recent past. Our results contribute toward the understanding of the evolutionary history of lions and complement conservation efforts to protect the diversity of this vulnerable species.

Comparative transcriptomic analysis reveals candidate genes for seasonal breeding in the male Lion-Head goose.

1. Due to seasonal breeding, geese breeds from Southern China have low egg yield. The genetic makeup underlying performance of local breeds is largely unknown, and few studies have investigated this problem. This study integrated 21 newly generated and 50 publicly existing RNA-seq libraries, representing the hypothalamus, pituitary and testis, to identify candidate genes and importantly related pathways associated with seasonal breeding in male Lion-Head geese.2. In total, 19, 119 and 302 differentially expressed genes (DEGs) were detected in the hypothalamus, pituitary and testis, respectively, of male Lion-Head geese between non-breeding and breeding periods. These genes were significantly involved in the neuropeptide signalling pathway, gland development, neuroactive ligand-receptor interaction, JAK-STAT signalling pathway, cAMP signalling pathway, PI3K-Akt signalling pathway and Foxo signalling pathway.3. By integrating another 50 RNA-seq samples 4, 18 and 40 promising DEGs were confirmed in hypothalamus, pituitary and testis, respectively.4. HOX genes were identified as having important roles in the development of testis between non-breeding and breeding periods of male Lion-Head geese.

Whole genome sequencing and the application of a SNP panel reveal primary evolutionary lineages and genomic variation in the lion (Panthera leo).

BACKGROUND: Previous phylogeographic studies of the lion (Panthera leo) have improved our insight into the distribution of genetic variation, as well as a revised taxonomy which now recognizes a northern (Panthera leo leo) and a southern (Panthera leo melanochaita) subspecies. However, existing whole range phylogeographic studies on lions either consist of very limited numbers of samples, or are focused on mitochondrial DNA and/or a limited set of microsatellites. The geographic extent of genetic lineages and their phylogenetic relationships remain uncertain, clouded by massive sampling gaps, sex-biased dispersal and incomplete lineage sorting. RESULTS: In this study we present results of low depth whole genome sequencing and subsequent variant calling in ten lions sampled throughout the geographic range, resulting in the discovery of >150,000 Single Nucleotide Polymorphisms (SNPs). Phylogenetic analyses revealed the same basal split between northern and southern populations, as well as four population clusters on a more local scale. Further, we designed a SNP panel, including 125 autosomal and 14 mitochondrial SNPs, which was tested on >200 lions from across their range. Results allow us to assign individuals to one of these four major clades (West & Central Africa, India, East Africa, or Southern Africa) and delineate these clades in more detail. CONCLUSIONS: The results presented here, particularly the validated SNP panel, have important applications, not only for studying populations on a local geographic scale, but also for tracing samples of unknown origin for forensic purposes, and for guiding conservation management of ex situ populations. Thus, these genomic resources not only contribute to our understanding of the evolutionary history of the lion, but may also play a crucial role in conservation efforts aimed at protecting the species in its full diversity.

Spatiotemporal Genetic Diversity of Lions Reveals the Influence of Habitat Fragmentation across Africa.

Direct comparisons between historical and contemporary populations allow for detecting changes in genetic diversity through time and assessment of the impact of habitat fragmentation. Here, we determined the genetic architecture of both historical and modern lions to document changes in genetic diversity over the last century. We surveyed microsatellite and mitochondrial genome variation from 143 high-quality museum specimens of known provenance, allowing us to directly compare this information with data from several recently published nuclear and mitochondrial studies. Our results provide evidence for male-mediated gene flow and recent isolation of local subpopulations, likely due to habitat fragmentation. Nuclear markers showed a significant decrease in genetic diversity from the historical (HE = 0.833) to the modern (HE = 0.796) populations, whereas mitochondrial genetic diversity was maintained (Hd = 0.98 for both). Although the historical population appears to have been panmictic based on nDNA data, hierarchical structure analysis identified four tiers of genetic structure in modern populations and was able to detect most sampling locations. Mitogenome analyses identified four clusters: Southern, Mixed, Eastern, and Western and were consistent between modern and historically sampled haplotypes. Within the last century, habitat fragmentation caused lion subpopulations to become more geographically isolated as human expansion changed the African landscape. This resulted in an increase in fine-scale nuclear genetic structure and loss of genetic diversity as lion subpopulations became more differentiated, whereas mitochondrial structure and diversity were maintained over time.

Microsatellite characterization and development of unified STR panel for big cats in captivity: a case study from a Seoul Grand Park Zoo, Republic of Korea.

The zoos manage small populations of endangered big cat species like tiger, lion, and leopard for display, research, and conservation breeding. Genetic management of these populations is essential to ensure long term survival and conservation utility. Here we propose a simple and cost effective microsatellite based protocol for the genetic management of captive big cats. We sampled 36 big cat individuals from Seoul Grand Park Zoo (Republic of Korea) and amplified 33 published microsatellite loci. Overall, allelic richness and gene diversity was found highest for leopards, followed by lions and tigers. Twelve of the thirty-three markers showed a high degree of polymorphism across all target species. These microsatellites provide a high degree of discrimination for tiger (1.45 × 10-8), lion (1.54 × 10-10), and leopard (1.88 × 10-12) and thus can be adopted for the genetic characterization of big cats in accredited zoos globally. During captive breeding, zoo authorities rely on pedigree records maintained in studbooks to ensure mating of genetically fit unrelated individuals. Several studies have reported errors in studbook records of big cat species. Microsatellites are simple and cost effective tool for DNA fingerprinting, estimation of genetic diversity, and paternity assessment. Our unified microsatellite panel (12-plex) for big cats is efficient and can easily be adopted by zoo authorities for regular population management.

Long live the king: chromosome-level assembly of the lion (Panthera leo) using linked-read, Hi-C, and long-read data.

BACKGROUND: The lion (Panthera leo) is one of the most popular and iconic feline species on the planet, yet in spite of its popularity, the last century has seen massive declines for lion populations worldwide. Genomic resources for endangered species represent an important way forward for the field of conservation, enabling high-resolution studies of demography, disease, and population dynamics. Here, we present a chromosome-level assembly from a captive African lion from the Exotic Feline Rescue Center (Center Point, IN) as a resource for current and subsequent genetic work of the sole social species of the Panthera clade. RESULTS: Our assembly is composed of 10x Genomics Chromium data, Dovetail Hi-C, and Oxford Nanopore long-read data. Synteny is highly conserved between the lion, other Panthera genomes, and the domestic cat. We find variability in the length of runs of homozygosity across lion genomes, indicating contrasting histories of recent and possibly intense inbreeding and bottleneck events. Demographic analyses reveal similar ancient histories across all individuals during the Pleistocene except the Asiatic lion, which shows a more rapid decline in population size. We show a substantial influence on the reference genome choice in the inference of demographic history and heterozygosity. CONCLUSIONS: We demonstrate that the choice of reference genome is important when comparing heterozygosity estimates across species and those inferred from different references should not be compared to each other. In addition, estimates of heterozygosity or the amount or length of runs of homozygosity should not be taken as reflective of a species, as these can differ substantially among individuals. This high-quality genome will greatly aid in the continuing research and conservation efforts for the lion, which is rapidly moving towards becoming a species in danger of extinction.

Harnessing genomics to trace the path of a viral outbreak in African lions.

Predicting the emergence of novel infectious diseases requires an understanding of how pathogens infect and efficiently spread in alternative naïve hosts. A pathogen's ability to adapt to a new host (i.e. host shift) oftentimes is constrained by host phylogeny, due to limits in the molecular mechanisms available to overcome host-specific immune defences (Longdon et al., 2014). Some pathogens, such as RNA viruses, however, have a propensity to jump hosts due to rapid mutation rates. For example, canine distemper virus (CDV) infects a broad range of terrestrial carnivores, as well as noncarnivore species worldwide, with a host range that is distributed across 5 orders and 22 families (Beineke et al., 2015). In 1993-1994, a severe CDV outbreak infected multiple carnivore host species in Serengeti National Park, causing widespread mortality and the subsequent decline of the African lion (Panthera leo) population (Roelke-Parker et al., 1996). While previous studies established domestic dogs (Canis lupus familiaris) as the disease reservoir, the precise route of transmission to lions remained a mystery, and a number of wild carnivore species could have facilitated viral evolution and spread. In this issue of Molecular Ecology, Weckworth et al. (2020) used whole-genome viral sequences obtained from four carnivore species during the CDV outbreak, in combination with epidemiological data, to illuminate the pathway and evolutionary mechanisms leading to disease emergence in Serengeti lions.

Genetic insights into dispersal distance and disperser fitness of African lions (Panthera leo) from the latitudinal extremes of the Kruger National Park, South Africa.

BACKGROUND: Female lions generally do not disperse far beyond their natal range, while males can disperse distances of over 200 km. However, in bush-like ecosystems dispersal distances less than 25 km are reported. Here, we investigate dispersal in lions sampled from the northern and southern extremes of Kruger National Park, a bush-like ecosystem in South Africa where bovine tuberculosis prevalence ranges from low to high across a north-south gradient. RESULTS: A total of 109 individuals sampled from 1998 to 2004 were typed using 11 microsatellite markers, and mitochondrial RS-3 gene sequences were generated for 28 of these individuals. Considerable north-south genetic differentiation was observed in both datasets. Dispersal was male-biased and generally further than 25 km, with long-distance male gene flow (75-200 km, detected for two individuals) confirming that male lions can travel large distances, even in bush-like ecosystems. In contrast, females generally did not disperse further than 20 km, with two distinctive RS-3 gene clusters for northern and southern females indicating no or rare long-distance female dispersal. However, dispersal rate for the predominantly non-territorial females from southern Kruger (fraction dispersers ≥0.68) was higher than previously reported. Of relevance was the below-average body condition of dispersers and their low presence in prides, suggesting low fitness. CONCLUSIONS: Large genetic differences between the two sampling localities, and low relatedness among males and high dispersal rates among females in the south, suggestive of unstable territory structure and high pride turnover, have potential implications for spread of diseases and the management of the Kruger lion population.

Genetic Evidence for Contrasting Wetland and Savannah Habitat Specializations in Different Populations of Lions (Panthera leo).

South-central Africa is characterized by an archipelago of wetlands, which has evolved in time and space since at least the Miocene, providing refugia for animal species during Pleistocene arid episodes. Their importance for biodiversity in the region is reflected in the evolution of a variety of specialist mammal and bird species, adapted to exploit these wetland habitats. Populations of lions (Panthera leo) across south-central and east Africa have contrasting signatures of mitochondrial DNA haplotypes and biparental nuclear DNA in wetland and savannah habitats, respectively, pointing to the evolution of distinct habitat preferences. This explains the absence of genetic admixture of populations from the Kalahari savannah of southwest Botswana and the Okavango wetland of northern Botswana, despite separation by only 500 km. We postulate that ancestral lions were wetland specialists and that the savannah lions evolved from populations that were isolated during arid Pleistocene episodes. Expansion of grasslands and the resultant increase in herbivore populations during mesic Pleistocene climatic episodes provided the stimulus for the rapid population expansion and diversification of the highly successful savannah lion specialists. Our model has important implications for lion conservation.

Odintifier--A computational method for identifying insertions of organellar origin from modern and ancient high-throughput sequencing data based on haplotype phasing.

BACKGROUND: Cellular organelles with genomes of their own (e.g. plastids and mitochondria) can pass genetic sequences to other organellar genomes within the cell in many species across the eukaryote phylogeny. The extent of the occurrence of these organellar-derived inserted sequences (odins) is still unknown, but if not accounted for in genomic and phylogenetic studies, they can be a source of error. However, if correctly identified, these inserted sequences can be used for evolutionary and comparative genomic studies. Although such insertions can be detected using various laboratory and bioinformatic strategies, there is currently no straightforward way to apply them as a standard organellar genome assembly on next-generation sequencing data. Furthermore, most current methods for identification of such insertions are unsuitable for use on non-model organisms or ancient DNA datasets. RESULTS: We present a bioinformatic method that uses phasing algorithms to reconstruct both source and inserted organelle sequences. The method was tested in different shotgun and organellar-enriched DNA high-throughput sequencing (HTS) datasets from ancient and modern samples. Specifically, we used datasets from lions (Panthera leo ssp. and Panthera leo leo) to characterize insertions from mitochondrial origin, and from common grapevine (Vitis vinifera) and bugle (Ajuga reptans) to characterize insertions derived from plastid genomes. Comparison of the results against other available organelle genome assembly methods demonstrated that our new method provides an improvement in the sequence assembly. CONCLUSION: Using datasets from a wide range of species and different levels of complexity we showed that our novel bioinformatic method based on phasing algorithms can be used to achieve the next two goals: i) reference-guided assembly of chloroplast/mitochondrial genomes from HTS data and ii) identification and simultaneous assembly of odins. This method represents the first application of haplotype phasing for automatic detection of odins and reference-based organellar genome assembly.

Revealing the maternal demographic history of Panthera leo using ancient DNA and a spatially explicit genealogical analysis.

BACKGROUND: Understanding the demographic history of a population is critical to conservation and to our broader understanding of evolutionary processes. For many tropical large mammals, however, this aim is confounded by the absence of fossil material and by the misleading signal obtained from genetic data of recently fragmented and isolated populations. This is particularly true for the lion which as a consequence of millennia of human persecution, has large gaps in its natural distribution and several recently extinct populations. RESULTS: We sequenced mitochondrial DNA from museum-preserved individuals, including the extinct Barbary lion (Panthera leo leo) and Iranian lion (P. l. persica), as well as lions from West and Central Africa. We added these to a broader sample of lion sequences, resulting in a data set spanning the historical range of lions. Our Bayesian phylogeographical analyses provide evidence for highly supported, reciprocally monophyletic lion clades. Using a molecular clock, we estimated that recent lion lineages began to diverge in the Late Pleistocene. Expanding equatorial rainforest probably separated lions in South and East Africa from other populations. West African lions then expanded into Central Africa during periods of rainforest contraction. Lastly, we found evidence of two separate incursions into Asia from North Africa, first into India and later into the Middle East. CONCLUSIONS: We have identified deep, well-supported splits within the mitochondrial phylogeny of African lions, arguing for recognition of some regional populations as worthy of independent conservation. More morphological and nuclear DNA data are now needed to test these subdivisions.

Development of MHC-Linked Microsatellite Markers in the Domestic Cat and Their Use to Evaluate MHC Diversity in Domestic Cats, Cheetahs, and Gir Lions.

Diversity within the major histocompatibility complex (MHC) reflects the immunological fitness of a population. MHC-linked microsatellite markers provide a simple and an inexpensive method for studying MHC diversity in large-scale studies. We have developed 6 MHC-linked microsatellite markers in the domestic cat and used these, in conjunction with 5 neutral microsatellites, to assess MHC diversity in domestic mixed breed (n = 129) and purebred Burmese (n = 61) cat populations in Australia. The MHC of outbred Australian cats is polymorphic (average allelic richness = 8.52), whereas the Burmese population has significantly lower MHC diversity (average allelic richness = 6.81; P < 0.01). The MHC-linked microsatellites along with MHC cloning and sequencing demonstrated moderate MHC diversity in cheetahs (n = 13) and extremely low diversity in Gir lions (n = 13). Our MHC-linked microsatellite markers have potential future use in diversity and disease studies in other populations and breeds of cats as well as in wild felid species.

Evaluation of microsatellite markers for populations studies and forensic identification of African lions (Panthera leo).

The South African lion (Panthera leo) population is highly fragmented. One-third of its wild lions occur in small (<1000 km(2)) reserves. These lions were reintroduced from other areas of the species' historical range. Management practices on these reserves have not prioritized genetic provenance or heterozygosity. These trends potentially constrain the conservation value of these lions. To ensure the best management and long-term survival of these subpopulations as a viable collective population, the provenance and current genetic diversity must be described. Concurrently, poaching of lions to supply a growing market for lion bones in Asia may become a serious conservation challenge in the future. Having a standardized, validated method for matching confiscated lion parts with carcasses will be a key tool in investigating these crimes. We evaluated 28 microsatellites in the African lion using samples from 18 small reserves and 1 captive facility in South Africa, two conservancies in Zimbabwe, and Kruger National and Kgalagadi Transfrontier Parks to determine the loci most suited for population management and forensic genetic applications. Twelve microsatellite loci with a match probability of 1.1×10(-5) between siblings were identified for forensics. A further 10 could be added for population genetics studies.

The Addis Ababa Lions: Whole-Genome Sequencing of a Rare and Precious Population.

Lions are widely known as charismatic predators that once roamed across the globe, but their populations have been greatly affected by environmental factors and human activities over the last 150 yr. Of particular interest is the Addis Ababa lion population, which has been maintained in captivity at around 20 individuals for over 75 yr, while many wild African lion populations have become extinct. In order to understand the molecular features of this unique population, we conducted a whole-genome sequencing study on 15 Addis Ababa lions and detected 4.5 million distinct genomic variants compared with the reference African lion genome. Using functional annotation, we identified several genes with mutations that potentially impact various traits such as mane color, body size, reproduction, gastrointestinal functions, cardiovascular processes, and sensory perception. These findings offer valuable insights into the genetics of this threatened lion population.

The genetic status and rescue measure for a geographically isolated population of Amur tigers.

The Amur tiger is currently confronted with challenges of anthropogenic development, leading to its population becoming fragmented into two geographically isolated groups: smaller and larger ones. Small and isolated populations frequently face a greater extinction risk, yet the small tiger population's genetic status and survival potential have not been assessed. Here, a total of 210 samples of suspected Amur tiger feces were collected from this small population, and the genetic background and population survival potentials were assessed by using 14 microsatellite loci. Our results demonstrated that the mean number of alleles in all loci was 3.7 and expected heterozygosity was 0.6, indicating a comparatively lower level of population genetic diversity compared to previously reported studies on other subspecies. The genetic estimates of effective population size (Ne) and the Ne/N ratio were merely 7.6 and 0.152, respectively, representing lower values in comparison to the Amur tiger population in Sikhote-Alin (the larger group). However, multiple methods have indicated the possibility of genetic divergence within our isolated population under study. Meanwhile, the maximum kinship recorded was 0.441, and the mean inbreeding coefficient stood at 0.0868, both of which are higher than those observed in other endangered species, such as the African lion and the grey wolf. Additionally, we have identified a significant risk of future extinction if the lethal equivalents were to reach 6.26, which is higher than that of other large carnivores. Further, our simulation results indicated that an increase in the number of breeding females would enhance the prospects of this population. In summary, our findings provide a critical theoretical basis for further bailout strategies concerning Amur tigers.

A molecular analysis of African lion (Panthera leo) mating structure and extra-group paternity in Etosha National Park.

The recent incorporation of molecular methods into analyses of social and mating systems has provided evidence that mating patterns often differ from those predicted by group social organization. Based on field studies and paternity analyses at a limited number of sites, African lions are predicted to exhibit a strict within-pride mating system. Extra-group paternity has not been previously reported in African lions; however, observations of extra-group associations among lions inhabiting Etosha National Park in Namibia suggest deviation from the predicted within-pride mating pattern. We analysed variation in 14 microsatellite loci in a population of 164 African lions in Etosha National Park. Genetic analysis was coupled with demographic and observational data to examine pride structure, relatedness and extra-group paternity (EGP). EGP was found to occur in 57% of prides where paternity was analysed (n = 7), and the overall rate of EGP in this population was 41% (n = 34). Group sex ratio had a significant effect on the occurrence of EGP (P < 0.05), indicating that variation in pride-level social structure may explain intergroup variation in EGP. Prides with a lower male-to-female ratio were significantly more likely to experience EGP in this population. The results of this study challenge the current models of African lion mating systems and provide evidence that social structure may not reflect breeding structure in some social mammals.

A new multiplex qPCR assay to detect and differentiate big cat species in the illegal wildlife trade.

All species of big cats, including tigers, cheetahs, leopards, lions, snow leopards, and jaguars, are protected under the Convention on the International Trade in Endangered Species (CITES). This is due in large part to population declines resulting from anthropogenic factors, especially poaching and the unregulated and illegal trade in pelts, bones, teeth and other products that are derived from these iconic species. To enhance and scale up monitoring for big cat products in this trade, we created a rapid multiplex qPCR test that can identify and differentiate DNA from tiger (Panthera tigris), cheetah (Acinonyx jubatus), leopard (Panthera pardus), lion (Panthera leo), snow leopard (Panthera uncia), and jaguar (Panthera onca) in wildlife products using melt curve analysis to identify each species by its unique melt peak temperature. Our results showed high PCR efficiency (> 90%), sensitivity (detection limit of 5 copies of DNA per PCR reaction) and specificity (no cross amplification between each of the 6 big cat species). When paired with a rapid (< 1 h) DNA extraction protocol that amplifies DNA from bone, teeth, and preserved skin, total test time is less than three hours. This test can be used as a screening method to improve our understanding of the scale and scope of the illegal trade in big cats and aid in the enforcement of international regulations that govern the trade in wildlife and wildlife products, both ultimately benefiting the conservation of these species worldwide.