McDowell and Graves: Nuclear and mitochondrial DNA markers for identification of istiopfiorid and xiphiid billfisfies 



543 



I 2 3 



Figure 5 



Larval billfish and its corresponding specific identification as a sailfish based on tlie BM32-2 

 locus. Lane 1 Dra I, lane 2. 1-kb plus DNA ladder (Gibco/BRL Life Technologies Inc., Bethesda 

 MDl, lane3, Ddc/. 



Chow (1994) was also unable to distinguish between the 

 two species based on RFLP analysis of the cytochrome b 

 gene, and Innes et al. ( 1998) did not consider white marlin 

 in their investigation because it does not occur in Austra- 

 lian waters. RFLP analysis of the whole mtDNA molecule 

 indicated that white and striped marlin share composite 

 haplotypes, although there are highly significant frequen- 

 cy differences between the species (Graves and McDowell, 

 1995; Graves, 1998). Sequence analysis of the mtDNA 

 cytochrome b gene also demonstrated a lack of genetic 

 divergence among white and striped marlin (Finnerty and 

 Block, 1995), and a further genetic analysis of the species' 

 relationships is warranted. 



To evaluate the utility of the methods outlined in our 

 study with those of other investigators, detailed protocols 

 and six unknown billfish samples were sent to the South- 

 east Fisheries Science Center's (now National Ocean 

 Survey's) Charleston, SC, laboratory. Scientists at the 

 Charleston Laboratory analyzed both mitochondrial and 

 nuclear DNA markers for each sample and arrived at con- 

 sistent, correct identifications for each of the samples of 

 unknown billfish. In addition, samples of juvenile billfish 

 collected by investigators at the University of Miami were 

 analyzed in our laboratory with these molecular markers. 

 Samples consisting of one eye taken from a 3-mm juvenile 

 billfish provided sufficient DNA to amplify the mtDNA 

 and nuclear markers, allowing specific identification 

 (Fig. 5). The technique is currently being used to deter- 

 mine the temporal occurrence of istiophorid larvae in the 

 Florida Straits iLuthy and McDowell'-). 



Although the methods presented in our study allow the 

 specific identification of billfish species, more sensitive mo- 



lecular markers are required to distinguish among ocean 

 populations of some istiophorid species. Amendment I to the 

 Fishery Management Plan for Atlantic Billfishes prohibits 

 the sale of blue marlin, white marlin, and sailfish taken in 

 the Atlantic Ocean, although it is legal to market blue mar- 

 lin, striped marlin, and sailfish from the Indian or Pacific 

 oceans. Enforcement of this regulation requires the ability 

 to discriminate between Atlantic and Indo-Pacific individu- 

 als of blue marlin, sailfish, and white and striped marlin. 

 Examination of our results suggests that there are several 

 other molecular markers, which while not used in this study, 

 occur at relatively high frequencies in Atlantic blue marlin, 

 sailfish, and white marlin but do not occur in their Pacific 

 conspecifics. These molecular markers could potentially be 

 used to identify some Atlantic individuals without misclas- 

 sifying a Pacific fish, thereby allowing the enforcement of 

 the management plan. Additional work will be required to 

 develop a database that would support such analyses. 



Acknowledgments 



We thank all those who so generously helped with collec- 

 tions including Julian Pepperell, Guy Harvey, Ed Everett, 

 Eric Prince, and Mike Judge, as well as the anglers at 

 Hotel Spa Buenavista, Baja California Sur, Mexico, and 

 The Mid-Atlantic 500,000 Tournament, Cape May, New 

 Jersey. We also thank Cheryl Woodley and the National 

 Ocean Services' Charleston Laboratory for helping to vali- 

 date our results. This project was funded by the National 

 Oceanic and Atmospheric Association's Saltonstall-Ken- 

 nedy Program grant number NA67FD00038. 



2 Luthy, S., and J. McDowell. 2001. A molecular approach to 

 the identification of larval billfishes. Submitted to the Austra- 

 lian Journal of Marine and Freshwater Research as part of the 

 y^ international billfish symposium. CSIRO Publishing, 150 

 Oxford St. Collingwood, Vic. 3066, Australia. 



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