Corvidae in Pakistan Are Represented by Two Distinct Clades Revealed Through Maternally Inherited Gene Region

Corvidae is a species rich and morphologically diverse family of the order Passeriformes (Aves), generally well identified by barcodes globally. Species identification and phylogenetic analysis through DNA barcodes using mitochondrial COI gene (cytochrome c oxidase subunit I) was aimed for samples of birds collected from different regions of Pakistan. Mitochondrial DNA was successfully extracted from keel tissue and Folmer region of COI gene comprising similar to 650 bps was amplified using universal primers and PCR products were confirmed by 1% agarose gel electrophoresis. Sequencing was carried out by Sanger's method and BLAST analysis identified these samples as five species (Urocissa flavirostris, Dendrocitta vagabunda, Corvus splendens, Corvus corax and Corvus macrorhynchos) from three genera of family Corvidae. The nucleotide sequences were submitted to the Barcode of Life Data System (BOLD) and the eligible sequences were assigned the Barcode Index Numbers (BINs). The COI gene sequence data of 8 species of family Corvidae were retrieved from GenBank (NCBI) database and phylogenetic relationship was established in these 13 barcodes. K2-parameter distances were calculated on MEGA7 software with overall average distance of 0.141 calculated at 1000 bootstrap repetitions. Phylogenetic tree reconstructed by neighbour joining method discriminated all species into two distinct clades initially and then into subclades based on similarity and variations in their sequences. Species of the same genera were grouped into one clade as genus Corvus. DNA barcoding was proved to be an effective tool for species molecular identification and their phylogenetic analysis during this study which may help in identification and biogeographic studies of birds in future.