444 



Fisher/ Bulletin 101(2) 



45" 



40" 



35" 



30° 



Figure 1 



Sampling locations for young-of-the-ycar (YOY) weakfish 

 iCynoscion regalis) in the summers of 1996 and 1997. Sites 

 are Peconic Bay, New York (NY); Delaware Bay, Delaware 

 (DB); Chesapeake Bay, Virginia (CB); Pamlico Sound, North 

 Carolina (NO; and Doboy Sound, Georgia (GA). 



sportfishes (including eight species of sciaenids) based on 

 a 12S/16S mtDNA gene region digested with Rsa I (Cordes 

 et al., 2001). Additional 12S/16S mtDNA/Rsa I patterns 

 were generated for silver seatrout iCynsoscion nothus) and 

 sand seatrout (C. arenarius) from the Gulf of Mexico, as 

 well as for banded drum {Larinius fasciatus), gulf kingfish 

 (Menticirrhus littoralis), and star drum iStellifer lanceola- 

 tus) from the South Atlantic Bight following procedures in 

 Cordes etal. (2001). 



The following microsatellite primers (Table 1 ) developed 

 for red drum (Sciaenops ocellatus) and spotted seatrout 

 {Cynoscion nehulosus) loci were used to amplify weakfish 

 DNA: SOC050 and SOC044 (Turner et al., 19981, SOC014 

 (Chapman'), and CNE612 (Chapman et al., 1999). Ampli- 

 fications of all microsatellite loci were carried out in 10 pL 



^ Chapman, R. W. 1998. Unpubl. data. Marine Resources Re- 

 search Institute, Department of Natural Resources, Charleston, 

 SC 29422. 



reactions containing 8.30 pL sterile dHgO, 1.0 pL lOx PCR 

 buffer with 15 niM MgCU, 0.20 pL 10 niM dNTP mixture, 

 0.05 pL forward primer ( 100 pm/pL) labeled with a fluores- 

 cent dye (Licor), 0.20 pL reverse primer (100 pm/pL), 0.05 

 pL Taq I polymerase (5 U/pL), and 0.20 pL weakfish DNA. 

 Samples were first denatured for 4 min at 95"C, followed by 

 32 cycles of PCR amplification performed under the follow- 

 ing conditions: 1 min. at 94°C, 1 min. at 50°C, and 1 min. 

 at 72°C. Reactions were given a final 7 min. extension at 

 72°C. PCR product alleles were separated electrophoreti- 

 cally on a 6% Long Ranger™' polyacrylamide gel with a 

 model 4000 automated DNA infrared sequencer from Li- 

 Cor (Lincoln, NE). 



Universal actin gene primers developed by G. Warr and 

 M.Wilson (cited in Reece et al., 1997) were used to identify 

 and refine an approximately 800-bp actin intron region lo- 

 cus (CRESIAl) in weakfish (Cordes, 2000). Weakfish PCR 

 amplification products obtained with S7 ribosomal protein 

 intron 2 (RP2) primers originally developed from swordfish 

 Xiphius gladius (Chow and Hazama, 1998) were cloned 

 and sequenced as described in Cordes (2000) and checked 

 against sequences published in Genbank to confirm their 

 identity. The original RP2 primers were then used without 

 modification for amplification of all samples. Both the CRE- 

 SIAl and RP2 amplifications were carried out under the 

 same conditions outlined above for the microsatellite loci, 

 with the exception that the annealing temperature was 

 lowered to 45°C. CRESIAl and RP2 amplification products 

 from a subset of each weakfish collection were screened 

 for polymorphisms with a panel of restriction endonucle- 

 ases and the resulting digestions were separated on 2.5% 

 agarose gels with 1% NuSieve and 1.5% agarose in IxTBE 

 buffer (Cordes, 2000). Gels were stained in IxTBE buffer 

 containing 30 pL (5 mg/niL) ethidium bromide (EtBri, vi- 

 sualized on a Spectroline model TR-302 transilluminator, 

 and photographed with a Polaroid CU-5 land camera. Dra I 

 was the only enzyme that revealed polymorphic restriction 

 sites within CRESIAl, and only Hinf I revealed reliably 

 scored polymorphisms in RP2. All YOY weakfish samples 

 were subsequently screened for variation at CRESIAI/Z)ra 

 IandRP2///in/'I. 



Microsatellite gel images and restriction enzyme diges- 

 tion patterns for CRESIAl and RP2 were analyzed by 

 using the software program RFLPScan Plus 3.0 (CSPI- 

 Scanalytics, 1996). Statistical analyses for all loci were 

 performed with the Arlequin 1.1 software program of 

 Schneider et al. (1997). Nonparametric, exact-significance 

 tests (exact significance tests and exact probability tests) 

 were used to evaluate sample genotype distributions for 

 departures from Hardy-Weinberg expectations. Unbiased 

 estimators of exact significance probabilities for the Hardy- 

 Weinberg equilibrium tests were calculated by using the 

 Markov chain algorithm of Guo and Thompson (1992) 

 with a Markov chain length of 100,000 steps. Patterns 

 of genetic diversity and divergence within and between 

 populations were evaluated by using the analysis of mo- 

 lecular variance (AMOVA) of Excoffier et al. (1992), which 

 generates F-statistics analogous to the Svaluesof Weir and 

 Cockerham (1984). Significance of /"'-statistics was evalu- 

 ated with exact F permutation procedures (Excoffier et al., 



