DNA barcode markers for birds of the genus Sporophila: advances in species identification

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BACKGROUND: Accurate identification of Sporophila is crucial for conservation and combating trafficking. Due to morphological similarity among females, molecular tools are essential. Although cytochrome c oxidase subunit 1 (COI) is the standard animal barcode, its limited resolution in recently diverged species necessitates more informative regions, such as cytochrome b (CYTB) and NADH dehydrogenase 2 (ND2), which are effective for Thraupidae. The aim of this study was to provide molecular mitochondrial markers to support species identification and development of tools that contribute to conservation of Sporophila. METHODS AND RESULTS: The markers were validated using 62 adult males (13 species) and applied to 47 unidentified females from the Centro de Triagem de Animais Silvestres (CETAS). DNA was amplified via PCR, targeting CYTB and ND2, using genus-specific primers for all samples. COI was amplified exclusively for males, using primers based on literature. Sequences were analyzed using an ABI3500.Maximum likelihood trees and haplotype networks compared each studied region. Nearest-neighbor and barcode gap analyses confirmed marker efficiency for species identification, with CYTB and COI demonstrating the highest reliability. Among the 13 species evaluated, five showed barcode gaps across all markers, while two depended on the marker used. Female samples achieved a 100% amplification rate, with 74.47% showing high identity (> 98%); conclusively, 52.13% were identified with high confidence, whereas the remainder belonged to species falling outside our markers' current discrimination efficiency. CONCLUSIONS: This study provides efficient tools for future research and conservation initiatives targeting Sporophila species.