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Abstract Small islands tend to lack predators because species at higher trophic levels often cannot survive. However, two exceptional top predators—the Iriomote cat Prionailurus bengalensis iriomotensis, and the Crested Serpent Eagle Spilornis cheela perplexus—live on the small Iriomote Island in the Ryukyu Archipelago. To understand how these predators coexist with limited resources, we focused on their seasonal diets between which conflicts are considered to occur. To compare the diets, we used DNA metabarcoding analysis of faecal samples. In the summer, we identified 16 unique prey items from Iriomote cat faecal samples and 15 unique prey items from Crested Serpent Eagle faecal samples. In the winter, we identified 37 and 14, respectively. Using a non-metric multidimensional scaling and a permutational multivariate analysis of variance, our study reveals significant differences in the diet composition at the order level between the predators during both seasons. Furthermore, although some prey items at the species-to-order level overlapped between them, the frequency of occurrence of most prey items differed in both seasons. These results suggest that this difference in diets is one of the reasons why the Iriomote cat and the Crested Serpent Eagle are able to coexist on such a small island.

Two subspecies of mainland leopard cats (Prionailurus bengalensis), Iriomote cats (Prionailurus bengalensis iriomotensis), and Tsushima leopard cats (Prionailurus bengalensis euptilurus), are small, endangered felids that are endemic to the Japanese islands, Iriomote-jima and Tsushima, respectively. Both subspecies have an estimated population size of only about 100 individuals each. Therefore, it is crucial to expand our understanding of these subspecies to successfully conserve them. While their morphology, anatomy, ecology, pathology, and genetics are well studied, little is known about their physiology regulated in subspecies-specific manners. This study characterized their urinary volatile and nonvolatile organic compounds, with which individuals express their physiological status and territorial ownership. We found significantly higher levels of 3-mercapto-3-methyl-1-butanol and its structural analogs, which are scent signals of domestic cats, in the urinary headspace of Iriomote cats than in that of Tsushima leopard cats. There were no differences in the urinary levels of felinine, an amino acid that acts as a precursor of 3-mercapto-3-methyl-1-butanol, suggesting the variation of catalytic activities for decomposing felinine to 3-mercapto-3-methyl-1-butanol between subspecies. Both subspecies exhibited physiological proteinuria due to the secretion of carboxylesterase 5A, also known as cauxin, for the production of felinine. In conclusion, while the two endangered Japanese subspecies of mainland leopard cats share similar genetic backgrounds, urinary contents of metabolites vary between the subspecies. We suggest that after the separation into two subspecies, the regulation of the metabolic pathway for 3-mercapto-3-methyl-1-butanol production likely evolved differently in each subspecies in response to their specific environments.