DNA Barcoding of Mammal Specimens Deposited in the Natural History Museum of Iran’s Department of Environment

Abstract

Reliable species identification is fundamental for biodiversity assessment and wildlife conservation. Here, we applied mitochondrial DNA barcoding to 90 unidentified mammalian specimens housed in the Natural History Museum of Iran’s Department of Environment using three mitochondrial markers, COI, D‑loop, and Cytb. COI sequences (643 bp) were successfully obtained from 55 specimens representing 23 species, while D‑loop (669 bp) and Cytb (755 bp) sequences were generated from 27 to 8 specimens, respectively, improving the identification of taxa unresolved by COI alone. Sequence similarity searches against GenBank references showed 96–100% identity values and enabled the identification of 45 mammalian species. Bayesian phylogenetic analyses recovered well‑supported species‑specific clusters, consistent with taxonomic assignments. Pairwise genetic distances estimated using the Kimura two‑parameter (K2P) model revealed clear interspecific divergence (> 2%) for most taxa, whereas closely related species exhibited comparatively low mitochondrial divergence. Our findings demonstrate the effectiveness of combining multiple mitochondrial markers for accurate species‑level identification of Iranian mammals and provide new reference sequences for future molecular, ecological, and conservation studies.