Phylogenetic analysis and BOLD Barcode Index Number clustering

To construct the phylogenetic tree, we downloaded the sequences of N. inferior, N. quadrimaculalis, and N. occultalis (two sequences, respectively) from GenBank. Patania ruralis was included as the outgroup because Patania (= Pleuroptya) is considered to be closely related to Nagiella based on male and female genitalia (e.g., Munroe 1976; Inoue 1982), but wing maculation, host plants, and the results of the phylogenetic analysis of Lu and Du (2020) suggest they are clearly different (see also Discussion for the differences between Patania and Nagiella). The sequences were aligned using MEGA 7.0 (Kumar et al. 2016). A neighbor-joining (NJ) tree was constructed using MEGA 7.0 based on Kimura 2-parameter model (Kimura 1980), and the bootstrap values were calculated with 1,000 replicates. Detection of variation sites and the number of intra/interspecific substitutions were calculated using MEGA 7.0.

DNA barcoding employs DNA sequences in a short and standardized gene region to facilitate species identification. BOLD (http://www.boldsystems.org/) is an international repository of DNA barcodes (Ratnasingham and Hebert 2007). The sequences in BOLD are clustered depending on their divergences and each cluster is given a unique Barcode Index Number (BIN) (Ratnasingham and Hebert 2013), an identifier for DNA barcode-based cluster corresponding to species. We searched the BOLD database for BINs that matched sequences obtained in this study.