LAUSR

Simple Summary Insecta is the most diverse group in the kingdom Animalia, and it accounts for approximately two-thirds of all animals. The most commonly used gene for species delimitation in animals is cytochrome c oxidase subunit 1 (COI). We calculated the genetic distance of 64,414 insect species, downloaded from BOLD, and found that approximately one-quarter of the species of Insecta showed high intraspecific genetic variation (>3%). Owing to the high intraspecific genetic variation in insects, false positives may easily occur in threshold-based species delimitation based on the COI gene. Compared to the fixed thresholds, the thresholds that were calculated from the “threshOpt” and “localMinima” algorithms that are embedded in the Spider package are recommended in threshold-based species delimitation studies. Abstract The genetic variation in the COI gene has had a great effect on the final results of species delimitation studies. However, little research has comprehensively investigated the genetic divergence in COI among Insecta. The fast-growing COI data in BOLD provide an opportunity for the comprehensive appraisal of the genetic variation in COI among Insecta. We calculated the K2P distance of 64,414 insect species downloaded from BOLD. The match ratios of the clustering analysis, based on different thresholds, were also compared among 4288 genera (35,068 species). The results indicate that approximately one-quarter of the species of Insecta showed high intraspecific genetic variation (>3%), and a conservative estimate of this proportion ranges from 12.05% to 22.58%. The application of empirical thresholds (e.g., 2% and 3%) in the clustering analysis may result in the overestimation of the species diversity. If the minimum interspecific genetic distance of the congeneric species is greater than or equal to 2%, it is possible to avoid overestimating the species diversity on the basis of the empirical thresholds. In comparison to the fixed thresholds, the “threshOpt” and “localMinima” algorithms are recommended for the provision of a reference threshold for threshold-based species delimitation studies.