DNA barcoding has emerged as a useful tool for the identification and discovery of animal species. It employs sequence diversity in a 648 base pair fragment near the 5′ end of the mitochondrial cytochrome c oxidase subunit I (COI) gene as a tool for species discrimination (Hebert et al. 2003a). Barcoding has been shown to discriminate species across the animal kingdom (Tyagi et al. 2010; Virgilio et al. 2010) including fishes, mammals, birds, insects, crustaceans and many other groups (Hebert et al. 2004a; Foottit et al. 2008; Hastings et al. 2008; Hubert et al. 2008; Hou et al. 2009; Wong et al. 2009; Clare et al. 2011). Reflecting the rapid growth in barcode coverage (Jinbo et al. 2011), BOLD, the Barcode of Life Data System (Ratnasingham & Hebert 2007), now includes records for more than 261K animal species. The order Lepidoptera has received particular attention (Hajibabaei et al. 2006; Silva-Brandao et al. 2009; Hebert et al. 2010; Kim et al. 2010) with 691K barcode records on BOLD (Feb 3, 2013), including data for 9124 named butterfly (Papilionoidea, Hesperioidea) species from 194 countries.
The gap between maximum intraspecific and minimum interspecific distances has been used for species delimitation in various animal groups (Hebert et al. 2004a; Meyer & Paulay 2005; Meier et al. 2006, 2008; Puillandre et al. 2012). This approach has helped to resolve cryptic species complexes (Hebert et al. 2004b; Burns et al. 2007; Park et al. 2011; Deng et al. 2012) and has aided ecological studies (Valentini et al. 2009; Pramual & Kuvangkadilok 2012). For example, Vaglia et al. (2008) used DNA barcodes to reveal cryptic species of sphingid moths, while van Nieukerken et al. (2012) discriminated cryptic species of leaf-mining Lepidoptera. Likewise, Carletto et al. (2009) discriminated sibling species of Aphis gossypii.
The effectiveness of DNA barcoding has spurred efforts to construct DNA barcode reference libraries for various animal groups (Ekrem et al. 2007; Guralnick & Hill 2009; Janzen et al. 2009; Lee et al. 2011; Zhou et al. 2011; Webb et al. 2012). These libraries not only aid the documentation of biodiversity (Janzen et al. 2005; Naro-Maciel et al. 2010) including endangered species (Elmeer et al. 2012; Vanhaecke et al. 2012), but can disclose endemism (Bossuyt et al. 2004; Quilang et al. 2011; Sourakov & Zakharov 2011). Because Lepidoptera have been selected as a model group for intensive analysis, the order is well represented on BOLD, but some regions such as South-East Asia have seen little investigation. Barcode records are available for a significant fraction of the Central Asian butterfly fauna (Lukhtanov et al. 2009) and for a smaller number of species from Western India (Gaikwad et al. 2012). However, these studies fail to provide coverage for many species known from Pakistan (Roberts 2001). The current study had the primary goals of testing the effectiveness of DNA barcodes in the identification of butterfly species from Pakistan and comparing these records with those from other regions to gain a better sense of the extent of intraspecific variation.