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 (H_o− 0.33, H_E− 0.35, and N_A− 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 cytochromebgene (846 bp),NADH-5gene (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.

Non-invasive genetic approaches, particularly using fecal samples, are commonly used to study endangered and elusive species, as they are easy to collect with minimal permission and cause little disturbance to the subject population. However, such studies face limitations due to poor DNA yield, which affects the overall utilization of collected samples and increases data errors. Here, we evaluated the impact of sample age and collection season on the performance of DNA extracted from feces (spraints) of the Eurasian otter (Lutra lutra), a semi-aquatic apex predator in South Korean freshwater ecosystems. We found that PCR amplification success rates decreased more rapidly in summer (79.3–58.2%) compared to winter (99.2–84.8%) with extended environmental exposure. Genotyping error rates were higher in samples collected during summer, with the rate of error increase over time being significantly greater in summer than in winter. The hot and humid South Korean summer fosters microbial growth and fecal degradation, which negatively impacts DNA yield, reducing PCR amplification success and increasing genotyping errors. We recommend collecting otter feces during winter for better DNA quality. If sampling in summer is unavoidable, it is crucial to collect fresh samples, which can be facilitated by conducting frequent surveys of latrine sites.

Background: The tiger population in Southwest Primorye is small and predominantly isolated from the main Sikhote-Alin population, which constitutes approximately 90% of the wild Amur tiger population. By 1996, this population declined to fewer than 10 individuals, but it has since grown and expanded into nearby habitats, now numbering over 50 individuals. Therefore, the regular genetic monitoring of this population is essential, as it has grown from a few founding members and remained geographically isolated. Methods and results: Genetic diversity was assessed using nine heterologous microsatellite markers amplified from non-invasively collected samples of 20 individual tigers. The Southwest Primorye tiger population exhibited moderate genetic diversity, with allelic richness (Na) at 3.67 and observed heterozygosity (Ho) at 0.63. Additionally, we detected a slight tendency toward heterozygosity excess at several loci, with an overall negative FIS, which may be influenced by recent genetic admixture or subtle population structuring. comparative assessment between our study and Sugimoto et al. (2012) revealed a marginal increase in genetic diversity over time, suggesting improved genetic health of the population, potentially due to genetic exchange with other populations. Conclusions: The significant growth and expansion of the Southwest Primorye tiger population into adjacent areas of Northeast China over the past two decades suggest a positive population trajectory. This modest increase in genetic diversity indicates a potentially favorable population condition. However, continuous genetic monitoring remains essential to track genetic trends, detect potential risks, and inform conservation strategies. This study highlights the need for ongoing evaluations to ensure the long-term survival of the Amur tiger population in Southwest Primorye.