BULLETIN OF MARINE SCIENCE, 80(2): 353–367, 2007 353 Bulletin of Marine Science © 2007 Rosenstiel School of Marine and Atmospheric Science of the University of Miami SPECIFIC IDENTIFICATION OF WESTERN ATLANTIC OCEAN SCOMBRIDS USING MITOCHONDRIAL DNA CYTOCHROME C OXIDASE SUBUNIT I (COI) GENE REGION SEQUENCES Melissa A. Paine, Jan R. McDowell, and John E. Graves ABSTRACT Identification of scombrids (tunas, mackerels, bonitos, etc.) is difficult when morphological characters are ambiguous or missing, such as with early life history stages or tissues found in the stomachs of predators. e mitochondrial cytochrome c oxidase subunit I (COI) gene region was evaluated as a molecular marker for the specific identification of the 17 members of the family Scombridae common to the western Atlantic Ocean. A 950 base pair region in the COI gene was sequenced from up to 20 individuals of each species, and suites of nucleotide polymorphisms that unambiguously distinguish among these scombrid species were identified. A shorter 250 base pair fragment of COI proved to be sufficient for species identifica- tion and was better suited for analyzing degraded tissue samples. Scombrid larvae collected in the Florida Straits and scombrid remains in the stomachs of large pe- lagic predators were used to demonstrate the utility of both the long and short COI fragments. Members of the family Scombridae (tunas, mackerels, bonitos, etc.) are important components of pelagic ecosystems, with several species supporting large commer- cial and recreational fisheries throughout the world’s oceans. Proper identification of these species at all life stages and in various conditions, even as degraded stomach contents, is essential to better understand early life history characteristics and eco- logical relationships in the pelagic ecosystem, and to enable effective management. In addition, specific identification of processed tissues or fillets is necessary for en- forcement of fisheries management regulations. While specific identification of adult scombrids is essentially unambiguous (Col- lette and Nauen, 1983), identification is problematic in situations where morphologi- cal characters are difficult to interpret (early life history stages) or missing (fillets, digested stomach contents). Identification of early life history stages of scombrids has been especially challenging. Scombrid eggs, larvae (especially those of the ge- nus unnus), and juveniles generally cannot be distinguished unambiguously based solely on morphology (Richards et al., 1990). Molecular markers can provide a means for positive identification when morpho- logical identification is uncertain. Various molecular markers have been used to identify fish eggs and larvae including allozymes (Morgan, 1975), polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) analysis (Daniel and Graves, 1994; McDowell and Graves, 2002), multiplex PCR (Rocha-Olivares, 1998; Hyde et al., 2005) and sequencing (Hare et al., 1994; Kirby and Reid, 2001; Perez et al., 2005). Many of these techniques have been used to identify scombrids. Allozymes have been successfully used to discriminate between early juveniles of bigeye tuna unnus obesus (Lowe, 1839) and yellowfin tuna unnus albacares (Bonnaterre, 1788) (Graves et al., 1988) as well as between adult Pacific northern bluefin tuna unnus thynnus orientalis Serventy, 1956 and southern bluefin tuna unnus maccoyii Castelnau, 1872 (Ward, 1995). Several studies have used PCR/