The non-coding "control region" of the mitochondrial genome was amplified using primers ProL2 5'-ctg ccc ttg gct ccc aaa gc-3' and PheCacaH2 5'-ctt agc atc ttc agt gcc at-3' complementary to regions of the flanking t-Pro and t-Phe tRNA genes, respectively. Direct sequencing of the amplified product revealed heteroplasmy in sharks due to both single substitutions and indel mutations. Length mutations in a homopolymeric tract of thymine residues proved particularly problematic, resulting in multiple peaks in sequence chromatograms; therefore, amplified D-loops were cloned using Invitrogen's TOPO cloning kit prior to sequencing, and 2-5 clones per individual sequenced in both directions using dye-labeled primers and a Licor 4200 sequencer. Of the 1145 base pairs there was on average 1.6 differences between haplotypes in most heteroplasmic individuals. However, we detected several cases in which haplotypes within individuals were more distantly related than haplotypes sampled between individuals. Nevertheless, levels of within individual nucleotide diversity were low (<0.1%) and did not confound interpretations of within and between population differentiation.

RFLP haplotypes were determined by digestion with HinF I and Alu I following manufacturer's recommendations and the fragments visualized on 1.5% agarose gels stained with ethidium broomide.