Orbacz and Gaffney: Genetic stucture of Tautoga onitts 



337 



more than twice as fast as fish from Rhode Island 

 during their first year of hfe. However, in a common- 

 garden experiment with fish from Rhode Island, Dela- 

 ware, and Virginia, Martin (1993) found no genetic 

 basis for the difference in growth rates. To date, there 

 have been no published genetic analyses of population 

 structure in this species. 



The purpose of this study was to evaluate patterns 

 of genetic variation in tautog across the primary 

 range of the species, by examining DNA sequence 

 variation in several mitochondrial and nuclear genes. 

 We used restriction fragment length polymorphism 

 (RFLP) analysis to examine several regions of the 

 mitochondrial genome amplified by the polymerase 

 chain reaction (PCR). These included portions of four 

 protein-coding genes (cytochrome 6, cytochrome c 

 oxidase subunit I, ATP synthetase subunit 6, and 

 NADH dehydrogenase subunit 2) and a segment 

 including the entire control region, tRNA'''^'^, and 

 part of the 12S rRNA gene. In addition, we used 

 denaturing gradient gel electrophoresis ( DGGE ) het- 

 eroduplex analysis to examine a nuclear gene intron 

 and portions of the mitochondrial cytochrome b and 

 cytochrome c oxidase subunit I genes. 



Materials and methods 



Sample collection and DNA extraction 



Juvenile tautog under age two were collected between 

 1991-1993 from three sites in the northern, middle. 



and southern portions of the tautog's primary range: 

 Narragansett Bay, Rhode Island; Delaware Bay, Del- 

 aware; and Chesapeake Bay, Virginia (for details 

 regarding collection techniques see Martin, 1993). 

 Fish were stored at -20°C. In addition, fin clips were 

 taken from adult fish collected from Delaware Bay 

 in 1996. 



Total DNA extracts were prepared from white 

 muscle tissue and fin clip samples from 24 fish from 

 each of the three sites by using a Puregene DNA iso- 

 lation kit (Gentra Systems, Inc., Minneapolis, MN) 

 following the protocol specified for animal tissue. 



Polymerase chain reaction amplification of DNA 



The polymerase chain reaction (PCR) was used to 

 amplify parts of five regions of the mitochondrial 

 genome: cytochrome c oxidase subunit I (COI), ATP 

 synthetase 6 ( ATPase 6 ), cytochrome b ( cyt b ), NADH 

 dehydrogenase subunit 2 (ND2), and the control 

 region (D-loop). In addition, intron 6 of the LDH-A 

 gene was amplified. Amplifications were performed 

 in a Perkin-Elmer 480 thermocycler under conditions 

 optimized for each primer pair. Primer sequences are 

 listed in Table 1. To create PCR products suitable for 

 use in DGGE, primers COlf-L, CBl-L ,and LDH849R 

 were modified through the addition of a 15 base pair 

 (bp) GC clamp (5'-CCCGCCGCCGCCGCC-3) to the 

 5' ends. 



An approximately 680-bp portion of the COI gene 

 was amplified with universal primers COla-H and 

 COlf-L (Palumbi et al., 1991) by using an initial 



