COI Gene-Based DNA Barcode Reference Database for Beetles in a Temperate Biodiversity Hotspot: Insights from the Liancheng Nature Reserve, Gansu Province, China

Beetles (Coleoptera) represent one of the most diverse insect groups and play vital ecological roles, yet their accurate identification is often challenging due to morphological similarities among taxa. DNA barcoding has emerged as a powerful and reliable tool for species-level identification and biodiversity monitoring. In this study, we established a local DNA barcode reference database for beetles in the Liancheng Nature Reserve, Gansu Province, China. From May to August 2024, beetle specimens were collected and identified using both morphological traits and DNA barcoding. Three species delimitation methods—Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), and Bayesian Poisson Tree Processes (bPTP)—were employed as complementary analytical tools, and phylogenetic relationships were inferred from cytochrome c oxidase subunit I (COI) sequences. A total of 164 COI sequences (650 bp) were obtained, representing 126 beetle species from 95 genera and 20 families. DNA barcoding successfully resolved morphologically ambiguous taxa, with many sequences reported here for the first time. Phylogenetic analysis revealed that species within the same genus formed cohesive clades before clustering at the family level, confirming the species-level discriminative power of the COI gene. Collectively, these findings demonstrate that COI-based DNA barcoding is a powerful complement to traditional taxonomy. The establishment of this preliminary reference database provides a valuable molecular resource for beetle identification and a practical tool to support biodiversity conservation, resource management, and long-term monitoring in the Liancheng Nature Reserve.