Identifying the Genetic Distance Threshold for Entiminae (Coleoptera: Curculionidae) Species Delimitation via COI Barcodes.

The subfamily Entiminae is the largest group in the family Curculionidae, and it has long represented a challenge in traditional and molecular classification. Here, we analyzed intra- and interspecific genetic distances of 621 public COI barcode sequences (658bp) from 39 genera and 110 species of Entiminae, to determine parameters most congruent in retaining established species. We found that the mean intraspecific genetic distance (3.07%) was much smaller than the mean interspecific one (21.96%), but there is a wide range of overlap between intra- and interspecific genetic distances (0.77-18.01%), indicating that there is no consistent, universal barcoding gap. Specifically, DNA barcoding gap analysis for morphospecies revealed that 102 of 110 morphospecies had barcoding gaps, and 9.18% was the optimum threshold of genetic distances for 97 species delimitation. We further confirmed this threshold with barcodes from 27 morphologically identified specimens (including 21 newly reported barcodes) sequenced from five genera and seven species. We also identified thresholds to delimit congeneric species within 14 selected genera (species > 2), which varied from 7.42% ( Trichalophus ) to 13.48% ( Barypeithes ). We herein present optimal parameters for species identification in the Entiminae. Our study suggests that despite no universal genetic distance threshold value in subfamily Entiminae, 9.18% is optimal for most species. We recommend a wider sampling of geographic populations to better account for intraspecific distance variation, and that genetic distance thresholds for species delimitation should be refined at the genus level.