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Analysis of mitochondrial DNA restriction fragment length polymorphism in European anchovy (Engraulis encrasicolus) revealed a large number of mitotypes that form two distinct clusters (phylads). Phylad A consists of one common mitotype and many rare secondary mitotypes that are one mutational step removed from the main type. Nucleotide diversity and number of homoplasious changes are low. Phylad B has a complex pattern of mitotype connectedness, high nucleotide diversity, and a large number of homoplasious changes. It is suggested that the two phylads evolved in isolation from each other and that present coexistence is the result of a secondary contact. Moreover, phylad A has a "star" phylogeny, which suggests that it has evolved in a population that experienced a drastic bottleneck followed by an explosion of size. Phylad A is practically the only phylad present in the Black Sea, with its frequency dropping to 85% in the northern Aegean, and to 40% in the rest of Mediterranean and the Bay of Biscay. The Black Sea is, therefore, the most likely place of origin of phylad A. Molecular data are consistent with a population bottleneck in the Black Sea during the last glaciation event and a subsequent exit of phylad A with the outflow into the Aegean following the ice melting. Phylogenetic analysis of anchovy mtDNA provides a reconstruction of population history in the Mediterranean, which is consistent with the geological information.

The Strait of Gibraltar has been proposed to be the divide between two marine biogeographical regions, the Mediterranean Sea and the Northeast Atlantic. Intraspecific studies have shown, for several of the examined species, a reduction of gene flow between the two basins. The present study examines genetic variation at nuclear and mitochondrial loci in five marine teleost species belonging to the family Sparidae. Four samples for each species were analysed spanning the Northeast Atlantic and the Mediterranean. For all individuals 17 allozyme loci were scored and a combined single strand conformation polymorphism‐sequencing approach was used to survey approximately 190 bp of the mitochondrial DNA (mtDNA) D‐loop region. All five species share similar biological features. For three species, namely Lithognathus mormyrus, Spondyliosoma cantharus, and Dentex dentex, large mtDNA divergence was observed between Atlantic and Mediterranean samples. Little or no mtDNA differentiation was found in the other two species, Pagrus pagrus and Pagellus bogaraveo. Allozyme data revealed strong differentiation when comparing Atlantic and Mediterranean samples of L. mormyrus and D. dentex, moderate for P. pagrus, and no differentiation for P. bogaraveo and S. cantharus. These results provide evidence for a sharp phylogeographical break (sensu Avise) between the Atlantic and the Mediterranean for two (or possibly three) sparid species of the five investigated. At the same time, the obtained results for the other two species raise the question on which ecological/historical factors might have caused the observed discrepancy in the geographical distribution of genetic variation among otherwise biologically similar species.

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BackgroundInternational fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection.Methodology/Principal FindingsThis study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology.Conclusions/SignificanceBased on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

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