A DNA barcode library for Korean Chironomidae (Insecta: Diptera) and indexes for defining barcode gap

Abstract

Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified larvae. The interspecies and intraspecies COI sequence variations were analyzed. Sophisticated indexes were developed in order to properly evaluate indistinct barcode gaps that are created by insufficient sampling on both the interspecies and intraspecies levels and by variable mutation rates across taxa. In a variety of insect datasets, these indexes were useful for re-evaluating large barcode datasets and for defining COI barcode gaps. The COI-based DNA barcode library will provide a rapid and reliable tool for the molecular identification of Korean chironomid species. Furthermore, this reverse-taxonomic approach will be improved by the continuous addition of other speceis' sequences to the library.

Fig. 1.

(A) Distribution and box plots of Kimura 2-parameter (K2P) genetic distance of mitochondrial cytochrome c oxidase I (COI) among 49 species. (B) Minimum interspecies and maximum intraspecies distances for COI gene regions were calculated by the K2P genetic distance model. The bottom region of the 1:1 line represents the presence of a DNA barcode gap. Diamonds (black) near the 1:1 line indicate controversial species (a: Cricotopus tricinctus; b: Cricotopus sylvestris; c: Chironomus sp. larva_3; d: Procladius choreus; e: Tanytarsus yoni; f: Dicrotendipes septemaculatus).

Fig. 2.

Neighbor-joining (NJ) tree of K2P distances based on mtDNA COI. The bootstrap value of 10,000 replicates is shown on each node.

Fig. 3.

Optimal cutoff value (Index_Cutoff), minimum interspecies distance (MinInter), and maximum intraspecies distance (MaxIntra) in the barcode datasets. These values are sorted by Index_Cutoff in ascending order.

Fig. 4.

Box plots depicting differences between the minimum interspecies variation (gray box plots) and the maximum intraspecies variation (white box plots) using the K2P distance model. The black dots indicate predicted cryptic species in each group.

Fig. 5.

Example of reverse taxonomy. Data include each clade in a NJ tree, bootstrap values on the branches, and the morphological characteristics of the adults [(a) head, (b) antennae, (c) wing, (d) abdomen, and (e) hypopygium] and larvae (gray box, showing the head capsule).