This study applied 18S rRNA metabarcoding to 13 fecal samples collected from the Japanese keelback (Hebius vibakari) on Jeju Island to identify its dietary composition and analyze its ecological characteristics. As expected, Next-generation sequencing (NGS)-based Relative Read Abundance (RRA) analysis revealed high dominance of the phylum Chordata (85.0%), largely attributed to host-derived DNA and genetically similar taxa. This pattern underscores a technical limitation of the 18S marker-its low taxonomic resolution for reptiles, which can lead to species-level misidentification. To address this limitation, we performed a complementary Frequencyof- Occurrence (FOO) analysis, which revealed that various invertebrate groups contributed substantially to the diet, including Araneae (spiders, 28.8%), Clitellata (earthworms, 21.2%), and Insecta (15.0%). Notably, the high occurrence of earthworms suggests that H. vibakari on Jeju Island may employ an active foraging strategy, probing moist soil layers rather than feeding exclusively on surface-dwelling insects. In conclusion, this study demonstrates that combining FOO analysis with 18S metabarcoding can overcome key limitations of the molecular approach and provides the first detailed characterization of the wild diet of H. vibakari on Jeju Island. Future studies incorporating host-blocking primers or additional genetic markers with higher taxonomic resolution are needed to further refine dietary inferences.

The objective of this study was to report the group mating of Asian Keelback snake, Hebius vibakari, in South Korea. On May 20, 2009, the group mating of H. vibakari was observed in the Maeng-gol do, Jindo-gun, Jeollanam-do, in the rocky area (107 m above sea level) on the ridge of the island. Six female and five male snakes made a form of mating behavior called “mating ball”. Two couples showed “tail-wrestling” which is the copulation behavior in snake species. During the mating, the temperature and humidity were 25.7°C and 59%, respectively. The result of this study was the first case report of the group mating in H. vibakari, a rare reptile.