Orbacz and Gaffney: Genetic stucture of Tautoga onttis 



339 



Table 2 



Frequencies of composite haplotypes from restriction digestion of the COI, cyt h, ATPase 6, ND2, and the control region amplified 

 fragments from T. onitis. Restriction enzymes used for each PCR product are listed. RI = Rhode Island, DE = Delaware, VA = 

 Virginia. All individuals had identical haplotypes for COI, cyt b, and ATPase 6 for the enzymes surveyed. 



COI: Hae III, Hinfl. NIa III, Dde I, Mnl I. Aci I and Tsp509 I 



cyt 6: Hae III. Hha I, aTaq I, NIa III. BstV I, Mse I. Dde I, Mnl 1, Aci 1, and 7sp509 I 



ATPase 6: Alu I, Rsa I, Hae III, Hha I. aTaq I, Hinfl. NIa III, BstV I, Mse I, Dde I, Mnl I, Ac, I, and Tsp509 I 



ND 2Alu I, Hoc III, Rsa I, Hha I, M,sp I. Hind, Dpn II, Ma III, BstV I, Mac I, DA I, Mnl I, Aci I and Tsp509 I 



control region: Alu I, iJsa I, //ae III, Hha I, oTaq I, Msp I, //(>if I, Dpn II, Ma III, BstV I, Mse I, Dde I, Mn/ I, Aci I and 7sp509 I 



Haplotype 



ND2 



Control region 



RI 



DE 



VA 



AAAAAAAAAAAAAA 

 AAAAABAAAAAAAA 

 AAAAAAAAAAAAAA 

 AAAAAAAAAAAAAA 



AAAAAAAAAAAAAAA 

 AAAAAAAAAAAAAAA 

 AABAAAAAAAAAAAA 

 AACAAAAAAAAAAAA 



eroduplex bands were considered to possess identi- 

 cal haplotypes. 



DNA sequencing 



Haplotypes identified by DGGE-heteroduplex screen- 

 ing of the cytochrome 6 fragment were sequenced by 

 using an ABI 373 automated DNA sequencer with 

 dye terminator chemistry. Sequencing was done in 

 both directions with the original PCR primers. 



Statistical analysis of RFLP data 



Restriction patterns were analyzed with the Restric- 

 tion Enzyme Analysis Package (REAP, version 4.0) 

 (McElroy et al., 1992). For each of the three geo- 

 graphical samples, haplotype diversities were calcu- 

 lated following Nei ( 1987), and nucleotide diversities 

 were calculated following Nei and Miller (1990). 

 Haplotype diversity ranges from zero (all individuals 

 share a common haplotype) to one (every individual 

 has a unique haplotype) and estimates the proba- 

 bility that two randomly selected individuals in a 

 sample will have different haplotypes. Nucleotide 

 diversity estimates the average number of nucleo- 

 tide substitutions for a pair of haplotypes randomly 

 drawn from a sample. Nucleotide divergences among 

 samples were estimated and corrected for within- 

 sample variation according to Nei ( 1987). Heteroge- 

 neity of haplotype frequencies across samples was 

 tested with exact R x C tests of independence using 

 the software program StatXact (Cytel, 1992). Addi- 

 tional analyses of population differentiation were 



conducted using Arlequin 1.1 (Schneider et al.^). 

 Power analysis of R x C tests was performed with 

 Power and Precision 1.0 (Borenstein et al., 1997). 



Results 



Restriction site variation 



Digestion with 16 restriction enzymes revealed poly- 

 morphisms in the ND2 and control region fragments, 

 but not in the COI, ATPase 6, or cyt b fragments. 

 Restriction enzyme digestion revealed an average of 

 129 restriction sites per individual and surveyed an 

 average of 532 bases in each fish. 



ND2 gene sequence variation was revealed by 

 digestion with Hini I, and sequence variation in 

 the control region fragment was revealed by diges- 

 tion with Hae III. The remaining enzymes produced 

 invariant patterns. The 16 enzymes revealed four 

 composite haplotypes attributable to restriction site 

 gains or losses in the 24 individuals surveyed 

 (Table 2). No additional haplotypes were uncovered 

 by increasing the sample size to 24 individuals from 

 each geogi'aphic region for the ND2-Hinfl and con- 

 trol region-Hae III combinations (Table 3). No evi- 

 dence of mitochondrial genome size variation or 

 heteroplasmy was observed in the regions analyzed. 



2 Schneider, S., J.-M. Kueffer, D. Roessli, and L. Excoffier 

 1997. Arlequin: a software for population genetic data anal- 

 ysis, vers. 1.1. Genetics and Biometry Lab., Department of 

 Anthropology, Univ. Geneva, Geneva. 



