McDowell and Graves: Nuclear and mitochondrial DNA markers for identification of istiopfiond and xiptniid blilfisfies 



541 



lation structure in blue marlin (Buonaccorsi et 

 al.. 1999). The short actin intron primers were 

 modified from "universal" actin gene primers 

 "480" and "483" (Siddall et al„ 2001). 



Both the short actin and the ITS marker were 

 rejected because neither marker amplified reli- 

 ably across species. For the scnDNA markers, 

 the program GeneJockey (Taylor, 1996) was 

 used to search for the presence of restriction 

 sites in sequences previously generated for blue 

 marlin. The WM08 marker was screened with 

 a total of ten enzymes, each of which produced 

 identical patterns across species. BM47 and 

 BM81 were also screened with ten enzymes 

 each. For BM47, the combination of enzymes 

 Bel I and Dele I appeared to be diagnostic in 

 a preliminary screening. However, upon further analy- 

 sis, it was discovered that this combination of locus and 

 enzymes produced confounding patterns for blue marlin 

 and sailfish; the most common pattern for blue marlin was 

 seen as a rare pattern for sailfish. Likewise, the BM81 lo- 

 cus did not distinguish between blue marlin and sailfish or 

 between white and striped marlin and spearfish with any 

 of the enzymes used. The MN32-2 locus was screened with 

 a total of nine restriction endonucleases. The combination 

 of Dra I and Dde I was found to allow for unambiguous 

 identification of billfish species (Figs. 3 and 4). 



As with the ND4 locus, after determining a diagnostic 

 enzyme-locus combination, we screened a total of 540 

 billfish samples from a broad geographic range to evalu- 

 ate the accuracy of the marker (Table 1 ). The enzyme Dra 

 I was found to have two alternate alleles: "D" and "E" for 

 blue marlin at a frequency of 19% and .5.5%, respectively. 

 All other species appeared to be fixed for different (ho- 

 mozygous) alleles with respect to this enzyme. For Dde 

 I, spearfishes had an alternate allele, "E," at a frequency 

 of 36%. In addition, blue marlin had two alternate alleles 

 "H" and "I" at frequencies of 16% and 40%, respectively 

 (Table 4). Although the "H" allele in blue marlin was the 

 only allele seen in black marlin, use of Dde I was not nec- 

 essary to distinguish the two species since they are easily 

 differentiated by Dra I (Figs. 3 and 4). All other species 

 appeared to be homozygous for different alleles. 



Discussion 



The purpose of this study was to develop a key to the iden- 

 tification of billfish species based on independent mito- 

 chondrial and nuclear markers. Our goal was to make the 

 process streamlined and capable of being performed in a 

 modestly equipped genetics laboratory. Specific identifica- 

 tion can be accomplished with a single PCR amplification 

 of either the mitochondrial ND4 locus or nuclear MN32-2 

 locus and two restriction digestions. Previous methods 

 with other mitochondrial gene regions have required the 

 use of either four or 13 restriction digestions (Innes et al., 

 1998 and Chow 1994, respectively). 



To facilitate specific identification, an objective of this 

 study, was to develop diagnostic markers that exhibited 



Table 4 



Restriction fragment patterns of the nuclear gene region 

 BM32-2 of istiophorid billfishes. This locus did not amplify 

 in the swordfish. 



(Al Digestions with Dra I. A = blue marlin; B = striped 

 marlin, white marlin, sailfish, black marlin; C = 

 spearfish; D = blue marlin; E = blue marlin. A/D 

 and A/E heterozygotes were seen in blue marlin cut 

 with Dra I. Individuals of all other species were fixed 

 homozygous. 



B 



C 



D 



1200 



(B) Digestions with Dde I. A = blue marlin; B = striped 

 marlin, white marlin; C = spearfish; D = sailfish; E = 

 spearfish; H = blue marlin; I = blue marlin. A/I A/H 

 and H/I heterozygotes were seen in blue marlin cut 

 with Dde I. C/E heterozygotes were seen in spearfish 

 cut with Dde I. Individuals of all other species were 

 fixed homozygous. 



limited intraspecific variation. Analysis of large sample 

 sizes (60-100 or more) of sailfish, white marlin, striped 

 marlin, blue marlin, and black marlin from throughout 

 each species' range revealed minimal variation of the spe- 

 cies-specific characters. Most species displayed a single 

 genotype for digestions with the two enzymes used to 

 cleave either the mitochondrial or nuclear amplification 

 products, and no species exhibited more than three geno- 

 types for any locus-restriction enzyme combination. In 



