DNA barcoding uncovered cryptic diversity in Seabob shrimp Xiphopenaeus kroyeri, Heller,1862 (Family: Penaeidae) from the South Mediterranean off Port Said coast, Egypt

This study investigated the phylogenetic relationship of Xiphopenaeus kroyeri in the Mediterranean and Atlantic regions. Total DNA was extracted from muscle tissue of frozen shrimp samples using the Tissue Extraction Kit (Qiagen). DNA integrity and quantity were assessed via agarose gel electrophoresis and NanoDrop. The COI gene was amplified using PCR with the LCO 1490 and HCO 2198 primer set. PCR reactions contained 12.5 µL 2x PCR Master Mix (GeneDireX), 1 µL of each primer (20 pmol), 2 µL DNA template (20 ng/µL), and 8.5 µL deionized water. Thermal cycling conditions included initial denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 45 sec, 53°C for 45 sec, and 72°C for 90 sec, with a final extension at 72°C for 7 min. COI products were purified using the QIAquick PCR Purification Kit (QIAGEN) and sequenced using a 3500 Genetic Analyzer (Applied Biosystems). Raw sequences were edited and uploaded to NCBI GenBank (accession numbers MW737452.1 and MW737453.1). Sequences were aligned using ClustalW, and a phylogenetic tree was constructed using the maximum likelihood method with 1000 bootstrap replicates under the Tamura 3-parameter substitution model. Portunus pelagicus COI sequences served as the outgroup. Phylogenetic analysis was performed using MEGA version 10.2.6. Phylogenetic analysis revealed a high degree of relatedness between the two successful X. kroyeri samples and those from the Atlantic-Pacific region. Two main COI haplotypes were identified, with the Egyptian samples belonging to one of them.