Identification of Cryptic Species and Haplotypes.

Cryptic species and haplotypes of rolled-leaf beetles were identified using the DNA barcode CO1. Over the past decade, numerous studies have demonstrated that this molecular marker can delimit species with extreme accuracy in many taxa (14). This DNA barcode is particularly effective in the identification of rolled-leaf beetle species. In previous studies, we were able to identify all morphologically distinct species of rolled-leaf beetles with 100% accuracy (14, 34). After measuring CT_max, we collected beetles in 95% (vol/vol) ET-OH. One leg was subsequently removed for DNA extractions following the protocols described by García-Robledo et al. (34). Sequences were aligned using MAFFT (35). DNA sequences were deposited in the GenBank (accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"KU357054-KU358485","start_term":"KU357054","end_term":"KU358485","start_term_id":"982859709","end_term_id":"982861142"}}KU357054-KU358485). A description of the methods used to delimit cryptic species and haplotypes is included in Fig. S3.