Genetic diversity of nine necrophagous Sarcophagidae (Diptera) species in Portugal.

Flesh flies (Diptera: Sarcophagidae) are among the most important insect groups used in forensic investigations, particularly for post-mortem interval estimation and assessing possible corpse relocation. Their practical use, however, is limited by the high morphological similarity of closely related species and by the limited representation of regional populations in DNA reference libraries. This study assessed two mitochondrial markers, cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 4 (ND4), for the identification and population-level characterisation of nine necrophagous Sarcophaga species collected in Portugal. A total of 41 adult male specimens were analysed to generate paired COI and ND4 sequences, which were deposited in GenBank. Both loci supported species-level separation, and haplotype networks and phylogenetic analyses revealed a consistent mitochondrial structure across the analysed taxa. Sarcophaga pandellei showed the highest intraspecific mitochondrial divergence, with one haplotype separated from the remaining conspecific haplotypes by almost 50 mutational steps and the highest nucleotide diversity of all species analysed. In contrast, S. africa and S. argyrostoma showed very low intraspecific variation. These findings support the forensic usefulness of COI and ND4 for identifying Portuguese necrophagous Sarcophaga, while also showing that mitochondrial evidence must be interpreted with attention to regional population structure and sampling limitations. The dataset fills an important gap for the Iberian Peninsula and strengthens integrative forensic identification in southern Europe.