Molecular identification of archaic bones as a native Korean black bear: implications for the ongoing bear restoration program.

The genetic investigation of the archeological or museum samples, including endangered species, provides vital information necessary to plan, implement, and revisit conservation strategies. In South Korea, the Asian black bear went almost extinct in wild by 2002, without leaving any authentic specimens representing the native population. Recently researchers found a set of animal bones in a natural cave in Mt. Taebaek (South Korea), suspected to be of a bear. In the present study, we undertook a molecular investigation and radiocarbon dating to establish the species' identity, phylogenetic position, and approximate age of the recovered specimen. The genetic investigation (CytB, COI, D-loop, SRY, and ZFX-ZFY) identified the sample as a male Asian black bear with close phylogenetic affinity with Northeast Asian bears. Radiocarbon dating estimated the bones to be aged 1800-1942 calAD. These findings indicate that the bone specimens found in the natural cave in Mt. Taebaek were from an individual that naturally inhabited South Korea long before the importing of farm bears (the 1980s) and initiation of wild population restoration (2004). The present study provides the first genetic information record of the native South Korean black bear. Our findings reaffirm the appropriateness of the ongoing bear restoration program in South Korea, with the reintroduction of individuals from North Korea and Russia.

Efficient and cost-effective non-invasive population monitoring as a method to assess the genetic diversity of the last remaining population of Amur leopard (Panthera pardus orientalis) in the Russia Far East.

Small populations of the endangered species are more vulnerable to extinction and hence require periodic genetic monitoring to establish and revisit the conservation strategies. The Amur leopard is critically endangered with about 100 individuals in the wild. In this study, we developed a simple and cost-effective noninvasive genetic monitoring protocol for Amur leopards. Also, we investigated the impact of fecal sample's age, storage, and collection season on microsatellite genotyping success and data quality. We identified 89 leopard scats out of the 342 fecal samples collected from Land of the Leopard between 2014-2019. Microsatellite genotyping using 12 markers optimized in 3 multiplex PCR reactions reveals presence of at least 24 leopard individuals (18 males and 6 females). There was a significant difference in the success rate of genotyping depending on the time from feces deposition to collection (p = 0.014, Fisher's exact test), with better genotyping success for samples having <2 weeks of environmental exposure. Amur leopard genetic diversity was found low (Ho- 0.33, HE- 0.35, and NA- 2.57) with no visible population substructure and recent bottleneck signature. Although a historical bottleneck footprint was observed. Mitochondrial DNA diversity was also found low with two haplotypes differing by a point mutation reported in 1,769 bp of investigated sequence covering parts of cytochrome b gene (846 bp), NADH-5 gene (611 bp) and control region (312 bp). We recommend periodic genetic monitoring of wild Amur leopards following the proposed methodology to achieve cost effectiveness and efficiency.

Whole genome survey of big cats (Genus: Panthera) identifies novel microsatellites of utility in conservation genetic study.

Big cats (Genus: Panthera) are among the most threatened mammal groups of the world, owing to hunting, habitat loss, and illegal transnational trade. Conservation genetic studies and effective curbs on poaching are important for the conservation of these charismatic apex predators. A limited number of microsatellite markers exists for Panthera species and researchers often cross-amplify domestic cat microsatellites to study these species. We conducted data mining of seven Panthera genome sequences to discover microsatellites for conservation genetic studies of four threatened big cat species. A total of 32 polymorphic microsatellite loci were identified in silico and tested with 152 big cats, and were found polymorphic in most of the tested species. We propose a set of 12 novel microsatellite markers for use in conservation genetics and wildlife forensic investigations of big cat species. Cumulatively, these markers have a high discriminatory power of one in a million for unrelated individuals and one in a thousand for siblings. Similar PCR conditions of these markers increase the prospects of achieving efficient multiplex PCR assays. This study is a pioneering attempt to synthesise genome wide microsatellite markers for big cats.

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.

Phylogenetic study of extirpated Korean leopard using mitochondrial DNA from an old skin specimen in South Korea.

The leopard, Panthera pardus, is a threatened species in its range throughout the world. Although, historically, the Korean Peninsula had a high population density of leopards, they were extirpated from South Korea by 1970, leaving almost no genetic specimens. Traditionally, Korean leopards are classified as Panthera pardus orientalis; however, their classification is based only on locality and morphology. Therefore, there is a need for genetic studies to identify the phylogenetic status of Korean leopards at the subspecies level. Presently, no extant wild specimen is available from South Korea; therefore, we extracted genetic material from the old skin of a leopard captured in Jirisan, South Korea in the 1930s and conducted the first phylogenetic study of the South Korean leopard. A total of 726 bp of mitochondrial DNA, including segments of the NADH5 and control region, were amplified by PCR. A phylogenetic analysis of the fragment, along with sequences of nine leopard subspecies from GenBank revealed that the extinct South Korean leopard belonged to the Asian leopard group and in the same clade as the Amur leopard (Panthera pardus orientalis). Thus, the leopard that inhabited South Korea in the past was of the same subspecies as the Amur leopard population currently inhabiting the transboundary region of Russia, China, and North Korea. These results emphasize the importance of conserving the endangered wild Amur leopard population (estimated to be about 60-80 individuals) in Russia and China, for future restoration of leopards in the Korean Peninsula.