Why restriction fragment length polymorphism analysis of mitochondrial DNA failed to resolve sardine (Sardinops) biogeography: insights from mitochondrial DNA cytochrome b sequences

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Abstract
Restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of mitochondrial DNA (mtDNA) have generated conflicting hypotheses on the phylogeography of Indian-Pacific clupeid Sardinops. An RFLP analysis of the control region and adjoining segments indicates a phylogenetic affiliation between southern Africa - Australian and Japanese sardines whereas comparisons of an overlapping segment of the control region sequence indicate an affiliation between southern Africa - Australian and Chile-Californian sardines. An analysis of 258 bp of mtDNA cytochrome b in 74 individuals of Sardinops from South Africa, Australia, Chile, California, and Japan revealed 24 haplotypes, resulting in high levels of haplotypic (h) diversity (average h = 0.70), low levels of nucleotide (pi) diversity (average pi = 0.005), and a satisfactory level of divergence (d) for population resolution (dmax = 4.02%). Cluster and parsimony analyses of haplotypic divergences confirmed the existence of three lineages corresponding to southern Africa - Australia, Chile-California, and Japan but failed to confirm a biogeographic connection between Australia and Japan. These results support the model based on control region sequences and highlight potential pitfalls of RFLP analyses of highly polymorphic DNA sequences with numerous homoplaseous transitions. A comparaison of 220 bp of cytochrome b in samples of Sardina and Sardinops produced an average sequence divergence of 23.2% and supports an ancient divergence between Sardina and Sardinops. The three lineages of Indian-Pacific Sardinops may merit subspecies recognition, based on low but significant levels of mtDNA divergence between them and on evidence of separations between these groups on the order of 200 000 - 500 000 years.

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