Greig et al.: Gene sequences useful for identification of western North Atlantic shark species 



519 



-80°C, dried, or stored in 70% EtOH. Blood was stored at 

 room temperature in sodium dodecyl sulfate-urea (SDS- 

 urea: 1% SDS, 8M urea, 240 mM Na 2 HP0 4 , ImM EDTA 

 pH 6.8). Total nucleic acids were extracted from frozen, 

 dried, and EtOH-preserved samples by using DNeasy 

 Tissue Kits and following manufacturer's recommenda- 

 tions (Qiagen, Valencia, CA). DNA was isolated from 

 blood in SDS-urea according to White and Densmore 

 (1992; protocol 11). Extracted DNA was visualized by 

 electrophoresis in a 1% agarose gel stained with 0.4 

 ng/mL of ethidium bromide in lx Tris-borate-EDTA 

 (TBE: 89 mM Tris-borate, 2 mM Na 2 EDTA, pH 8). A 

 1-kb DNA ladder (Promega, Madison. WD was used as 

 a size standard. 



Amplification and sequencing 



Primers 12SA-5' and 16SA-3' (Palumbi, 1996) were used 

 to amplify an approximately 1400-bp region spanning 

 the 3' end of the 12s rDNA, the valine tRNA, and the 

 5' end of the 16s rDNA region of mitochondrial DNA 

 (mtDNA). Samples were amplified in 50 uL reactions 

 containing -50 ng of template DNA, 20 mM Tris-HCl 

 pH 8.4, 50 mM KC1, 0.2 mM each dNTP, 2 mM MgCl 2 , 

 20 mM each primer, and 2.5 units Taq DNA polymerase 

 (Gibco BRL, Rockville, MD). Thermal cycling consisted of 

 an initial denaturation at 94°C for 1.5 minutes, followed 

 by 30 cycles of 40 seconds at 94°C, 40 seconds at 52°C, 

 and 50 seconds at 72°C, and a final extension step of 15 

 minutes at 72°C. Negative controls (no template) were 

 included in each set of reactions. PCR products were 

 gel-purified as described in Rosel and Block (1996) and 

 20-50 ng were used as template for ABI Big Dye Ter- 

 minator (v. 1.0, Applied Biosystems, Foster City, CA) 

 cycle sequencing reactions. Sequence was obtained with 

 amplification primers 12SA-5', 16SA-3' and two addi- 

 tional internal sequencing primers. Sequencing reaction 



products were precipitated with ethanol, washed accord- 

 ing to sequencing kit instructions, dried in a Savant 

 Speedvac Plus, and resuspended in 4 j<L of loading dye 

 (5:1 Hi-Di formamide:dextran blue). Fragments were 

 analyzed on an Applied Biosystems 377 automated DNA 

 sequencer. 



Sequence analysis and alignment 



Sequences were edited with SEQUENCHER (vers. 3.0; 

 Gene Codes Corp., Detroit, MI). We included three 

 additional sequences from GenBank: horn shark 

 (Heterodontus francisci, NC003137) to represent the 

 family Heterodontidae, thorny skate (Raja radiata, 

 AF106038), rabbit fish (Chimaera monstrosa, AJ310140), 

 and the Atlantic guitarfish {Rhhiobatis lentiginosus, 

 AY830717 — this study) to serve as outgroups for phy- 

 logenetic analyses. Sequences were aligned by using a 

 linear hidden Markov model (HMM) as implemented 

 in SAM (Sequence Alignment and Modeling System; 

 Hughey and Krogh, 1996; Karplus et al., 1998) with 

 default settings. The alignment file is available from 

 the senior author. 



Phylogenetic hypotheses were constructed by using 

 the maximum parsimony (MP) and neighbor-joining 

 (NJ) algorithms implemented in PAUP 4.0bl0 (Sinauer 

 Associates, Sunderland, MA). NJ analyses employed a 

 variety of pairwise distance measures, but the distance 

 measure used had little or no effect on the recovered 

 topologies. Phylogenies recovered with MP with equally 

 weighted characters were generally concordant with 

 those recovered by NJ, particularly when bootstrap 

 consensus trees were compared. For ease of interpre- 

 tation, we report NJ analyses using p-distances as a 

 metric. Bootstrapping (Felsenstein, 1985) was used to 

 estimate the reliability of NJ reconstructions (1000 

 pseudoreplicates). 



