Lankford et al : Population structure in Micropogonias undulatus 



885 



(RFLP ) were used 1 ) to document the magnitude and 

 spatial distribution of mtDNA variation in M. 

 undulatus from U.S. Atlantic and Gulf of Mexico lo- 

 calities, 2) to evaluate the integrity of Cape Hatteras, 

 North Carolina, as a genetic stock boundary, and 

 3) to estimate levels of gene flow among Atlantic lo- 

 calities to provide an improved basis for future deci- 

 sions regarding coastwide management of this fish- 

 ery resource. 



Methods 



Juvenile Atlantic croaker were collected from three 

 U.S. Atlantic estuaries (Delaware Bay, Cape Fear 

 River, and Indian River Lagoon) and one Gulf of 

 Mexico estuary (Louisiana) (Table 1). Sampling lo- 

 calities were chosen to represent the northern, cen- 

 tral, and southern portions of the species" U.S. At- 

 lantic coast range and to include areas north and 

 south of the hypothesized stock boundary at Cape 

 Hatteras, North Carolina. 



Genomic DNA extracts from individual croaker 

 were obtained by using the Puregene DNA isolation 

 kit (Centra Systems, Inc., Minneapolis. MN) and 

 were used in the polymerase chain reaction (Saiki et 

 al., 1988) to amplify two mtDNA regions: the pro- 

 tein-coding ATP synthetase subunit 6 gene (ATPase 

 6 ) and the noncoding "D-loop" segment of the control 

 region (Lankford, 1997). ATPase 6 was amplified by 

 using the primers ATPase 6-L and ATPase 6-H 

 (Quattro-^l. which yielded a 705-base-pair (bp) prod- 

 uct. Primers L-Thr (5-3': AGC TCA GCG CCA GAG 



^ Quattro, J. M. 1996. Department of Biological Sciences, Univ. 

 South Carolina. Personal comniun. 



CGCCGGTCTTGTAAA)and 12SAR-H (5-3: ATA 

 GTG GGG TAT CTA ATC CCA GTT) were used to 

 amplify a 1540-bp product containing the entire D- 

 loop region, tRNA-Pro and tRNA-Phe genes, and 

 portions of the 12S rRNA and tRNA-Thr genes (Lee 

 et al., 1995 ). Amplifications were performed in 50 ;uL 

 reaction volumes according to Kocher et al. (1989). 

 PCR products were digested with twelve restriction 

 endonucleases: Hinfi, HaelW, Mspl, Pvull, Sau96I, 

 Alul. Msel, /?,soI, Taql, Dclel, Nlalll, and Hhal (ob- 

 tained from New England BioLabs, Beverly, MA) 

 (Lankford, 1997). Variant RFLP patterns were con- 

 firmed by means of repeat digestions and electro- 

 phoresis on 39f NuSieve agarose gels. Fragment sizes 

 were estimated by using molecular weight markers: 

 pUC18-//at'III digest from Sigma-Aldrich Corp., St. 

 Louis, MO, pBR322-M.spI digest from New England 

 BioLabs, Beverly MA, and pBR322-Ss^Aa digest from 

 New England BioLabs: and the ANAGEL software 

 program (Mrazek and Spanova, 1992). 



Distinctive restriction-fragment patterns were 

 identified by letter codes and subsequently combined 

 to produce composite mtDNA haplotypes for indi- 

 vidual fish. Nucleon diversity (/; ) was calculated for 

 each locality (Nei and Tajima, 1981) with standard 

 errors estimated according to Nei ( 1987 ). Nucleotide 

 sequence diversity and nucleotide sequence diver- 

 gence were calculated with the Restriction Enzyme 

 Analysis Package (REAP, version 4.0) (McElroy et 

 al., 1992). Frequency distributions of composite 

 mtDNA haplotypes were tested for geogi'aphic homo- 

 geneity by using a Monte Carlo chi-square simulation 

 approach ( Roff and Bentzen, 1989) and a total of 10,000 

 random data resamplings. The integrity of Cape 

 Hatteras as a genetic stock boundaiy was tested by 

 comparing mtDNA haplotype frequencies north ( Dela- 



