537 



Abstract— I ndepondont molecular mar- 

 kers based on mitochondrial and nucle- 

 ar DNA were developed to provide pos- 

 itive identification of istiophorid and 

 xiphiid billfishes (marlins, spearfishes. 

 sailfish, and swordfish). Both classes 

 of markers were based on amplifica- 

 tion of short segments (<1.7 kb) of DNA 

 by the polymerase chain reaction and 

 subsequent digestion with informative 

 restriction endonucleases. Candidate 

 markers were evaluated for their abil- 

 ity to discriminate among the different 

 species and the level of intraspecific 

 variation they exhibited. The selected 

 markers require no more than two 

 restriction digestions to allow unam- 

 biguous identification, although it was 

 not possible to distinguish between 

 white marlin and striped marlin with 

 any of the genetic characters screened 

 in our study. Individuals collected from 

 throughout each species' range were 

 surveyed with the selected markers 

 demonstrating low levels of intraspecific 

 character variation within species. The 

 resulting keys provide two independent 

 means for the forensic identification of 

 fillets and for specific identification of 

 early life history stages. 



Nuclear and mitochondrial DNA markers for specific 

 identification of istiophorid and xiphiid billfishes^ 



Jan R. McDowell 



John E. Graves 



School of Marine Science 



Virginia Institute of Marine Science 



College of William and Mary 



Rt 1208 Create Rd 



Gloucester Point, Virginia 23062 



E mail address (for J McDowell), McDowell(q'VIM5 edu 



Manuscript accepted 18 September 2001. 

 Fish. Bull. 100:537-544 (2002). 



Species-level identification of most ma- 

 rine fishes is typically based on adult 

 characters. However, distinguishing char- 

 acters may be removed from adults 

 when they are processed for market or 

 personal consumption, making such 

 identification problematic. Furthermore, 

 species identification from early life 

 history stages of many marine fishes 

 is not possible because diagnostic mor- 

 phological characters at these stages 

 are not currently known. Consequently, 

 alternative means of identification are 

 needed. 



A variety of molecular genetic char- 

 acters have been used to provide iden- 

 tifications of marine fishes. Marine fish 

 eggs and larvae have been identified 

 by using allozymes (Mork, et al., 1983; 

 Graves et al., 1988), restriction analy- 

 sis of whole mitochondrial (mt) DNA 

 (Daniel and Graves, 1994), restriction 

 analysis of specific mtDNA gene regions 

 (Luczkovich et al.'), and specific ampli- 

 fication of mtDNA gene regions (Rocha- 

 Olivares, 1998 ). A similar suite of molec- 

 ular markers has been used to provide 

 positive identification of adult marine 

 fish tissues, with recent emphasis on 

 restriction analysis of amplified regions 

 of the mitochondrial genome (Chow et 

 al., 1993; Chow 1994, Chow and Kishino, 

 1995; Heist and Gold, 1997; Innes et al., 

 1998; Cordesetal., 2001). 



To be effective, a diagnostic molecular 

 marker must demonstrate consistent 

 differences among closely related spe- 

 cies and exhibit very limited intraspe- 

 cific variation. Restriction analyses of 

 regions of the mitochondrial genome 

 have met these criteria for several 

 marine fishes. However, reliance on a 

 single, maternally inherited character 



(mtDNA) can provide misleading results 

 in situations where there is a possibility 

 of hybridization or introgression, and 

 analyses of both nuclear and mitochon- 

 drial markers are therefore desirable. 



The istiophorid and xiphiid billfishes 

 (marlins, spearfishes, sailfish and 

 swordfish) represent an important 

 commercial and recreational fisheries 

 resource. Because of depleted stock 

 levels, current regulations within the 

 United States prohibit the sale of is- 

 tiophorid billfish taken in the Atlantic 

 Ocean. Although adult billfishes are 

 easily identified on the basis of morpho- 

 logical characters, these characters are 

 typically removed during processing, 

 preventing morphological identifica- 

 tion. In addition, the early life history 

 stages of istiophorid billfishes are not 

 well known, and specific identification 

 is problematic (Nakamura, 1985). 



Chow (1994) used 13 restriction en- 

 zymes in a restriction fragment length 

 polymorphism (RFLP) analysis of a 

 350-bp region of the mtDNA cytochrome 

 b gene to discriminate among ten nomi- 

 nal species of billfishes; however sam- 

 ples sizes were small for several species 



* Contribution 2470 of the Virginia Institute 

 of Marine Science, College of William and 

 Mary, Gloucester Point, VA 23062. 



' Luczkovich, J. J. ,H. J. Daniel, M.W. Sprague, 

 S. E. Johnson, R. C. PuUinger, T Jenkins, 

 and M.Hutchinson. 1999. Characteriza- 

 tion of critical spawning habitats of weak- 

 fish, spotted seatrout and red drum in Pam- 

 lico Sound using hydrophone surveys. Fi- 

 nal report to the North Carolina Division 

 of Marine Fisheries under grant numbers 

 F-62- 1 and F-62-2, 128 p. North Carolina 

 Department of Environment and Natural 

 Resources, Division of Marine Fisheries. 

 Morehead City, NC 28557. 



