Ángel S. Comesaña

Molecular Identification of Nine Commercial Flatfish Species by Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis of a Segment of the Cytochrome b Region

Commercial refrigerated or frozen flatfish fillets are sometimes mislabeled, and identification of these mislabeled products is necessary to prevent fraudulent substitution. Identification of nine commercial flatfish species (order Pleuronectiformes), Hippoglossus hippoglossus (halibut), Lepidorhombus boscii (four-spotted scaldfish), Lepidorhombus whiffiagonis (megrin), Platichthys flesus (flounder), Pleuronectes platessa (European plaice), Reinhardtius hippoglossoides (Greenland halibut), Scophthalmus maximus (turbot), Scophthalmus rhombus (brill), and Solea vulgaris (=Solea solea) (sole), was carried out on the basis of the amplification of a 486-bp segment of the mitochondrial genome (tRNA(Glu)/cytochrome b) by using the polymerase chain reaction (PCR) and universal primers. Sequences of PCR-amplified DNA from the flatfish species were used to select eight restriction enzymes (REs). The PCR products were cut with each RE, resulting in species-specific restriction fragment length polymorphism. Seven species groups could be identified by application of the single RE DdeI and six species groups by using HaeIII, HinfI, MaeI, or MboI. Different combinations of only a couple of these REs could unambiguously identify the nine flatfish species. Genetic polymorphisms of the target sequence were examined by comparison with previously published DNA sequences, and the results of this comparison confirmed the usefulness of this technique in distinguishing and genetically characterizing refrigerated or frozen pieces of these nine flatfish species.

Macrogeographic differentiation by mtDNA restriction site analysis in the S.W. European Mytilus galloprovincialis Lmk

The mussel Mytilus galloprovincialis Lmk. is distributed in Europe from the Mediterranean Sea to the English Channel and British Isles. Recently, an abrupt change at allozyme frequencies has been pointed out between Atlantic and Mediterranean Iberian populations. M. galloprovincialis samples on both sides of the allozyme discontinuity area were taken between 1992 and 1994. Four samples were from pure populations and one from the French hybrid zone of M. galloprovincialis and M. edulis L. A pure M. edulis sample was used as the control population. Mitochondrial DNA (mtDNA) restriction site analysis of EcoRI enzyme was applied. Significant differentiation at the haplotype frequencies between Atlantic and Mediterranean M. galloprovincialis populations was found, which represented a mean variation of about 27% for the FA haplotype frequencies. This mtDNA differentiation pattern was geographically concordant with that for allozyme loci previously reported. Moreover, significant differences at haplotype frequencies between M. galloprovincialis from the hybrid zone and from pure Atlantic populations were unexpectedly found, exhibiting a mean variation of about 27% for FA haplotype. FA frequencies for M. galloprovincialis from the hybrid zone (> 68%) were not intermediate between those for Atlantic pure populations (43%) and for M. edulis (< 25%). Evolutionary implications of these macrogeographic genetic differentiations are discussed.

ALLOZYME IDENTIFICATION OF MUSSELS (BIVALVIA: MYTILUS) ON THE PACIFIC COAST OF SOUTH AMERICA

Abstract The taxonomic identity of mussels in the southern hemisphere is still unclear, and the Mytilus that inhabit on the Pacific coast of South America has been considered by different authors as M. chilensis, M. edulis, M. edulis chilensis and M. galloprovincialis. To clarify the taxonomic identity of these mussels four samples from the northern limit of the distribution of Mytilus were taken as well as European control samples of M. edulis and M. galloprovincialis for comparison. Thirty allozyme loci were studied and 9 loci (Aco-1, Ap-1, Est-D, Gpi, Idh-1, Lap-1, Mpi, Me-2 and Odh) were partially diagnostic between the European M. edulis and M. galloprovincialis control samples, as previously reported. Chilean samples showed for four of the above partially diagnostic loci intermediate frequencies for typical alleles of M. edulis and M. galloprovincialis between those of the control samples, but they were closer to those of M. edulis for Ap-1 and Mpi and to those of M. galloprovincialis for Aco-1 and Est-D. The locus Lap-1 showed allele frequencies similar to those of M. edulis, whereas the most frequent allele of the loci Gpi, Idh-1 and Odh was that typical of M. galloprovincialis but at a higher frequency. Moreover, the partially diagnostic loci Me-2 and Lap-2, Pgm-2 and Pp showed important differences with regard to the control populations. Genetic distances, dendrograms and multidimentional scaling as well as principal component analysis on allele frequencies and factorial correspondence analysis on individual genotypes showed that South American samples were genetically closer to European M. galloprovincialis than to M. edulis but having particular and characteristic allele frequencies.

Mytilus galloprovincialis Lmk. in northern Africa

Mytilus edulis L. and M. galloprovincialis Lmk. are two forms of mussels that live and are cultivated on the Northeastern Atlantic coasts and where they coexist they hybridise in varying proportions. Up to date, the form or forms of Mytilus that inhabit the Atlantic coast of northern Africa have not been reliably determined. Four partially diagnostic allozyme loci for M. edulis and M. galloprovincialis (esterase-D, leucine aminopeptidase-1, mannose phosphate isomerase and octopine dehydrogenase) were investigated in mussel samples from five northern African sites in the Atlantic Ocean and the Alboran Sea. Pure European M. galloprovincialis and M. edulis samples were also studied for comparison. Evidence from diagnostic enzyme loci indicates that M. galloprovincialis occurs on the Atlantic and SW Mediterranean coasts of northern Africa, and no evidence for the occurrence of M. edulis, or hybrids between M. edulis and M. galloprovincialis, has been found in this area. The results are discussed in relation to the geographical distribution of both forms of Mytilus on Northeastern Atlantic coasts.

Identification of octopine dehydrogenase from Mytilus galloprovincialis

A cDNA encoding the putative octopine dehydrogenase (OcDH) from the mussel Mytilus galloprovincialis was cloned and sequenced. The complete coding region was expressed in the bacteria Escherichia coli and the recombinant protein was purified. The M. galloprovincialis OcDH appears to have the highest affinity for the amino acid substrate L-arginine (88.22%), compared to L-alanine (9.04%) and glycine (2.74%). This enzyme showed no activity when taurine or β-alanine was used as substrate. These data strongly support that this recombinant enzyme is octopine dehydrogenase and not another opine dehydrogenase such as alanopine or strombine dehydrogenases. The superimposition of the theoretical three-dimensional model of the M. galloprovincialis OcDH and the crystal structure of its homologous counterpart from the great scallop Pecten maximus showed interesting changes in the amino acid binding site which could explain the differences found in the substrate affinity between the two molluscs. A phylogenetic analysis was performed comparing M. galloprovincialis OcDH and annotated sequences representing the five opine dehydrogenase (OpDH) protein family members. The phylogenetic tree which was obtained clustered the OpDH enzymes according to the evolutionary relationships of the species and not to the biochemical reaction catalysed. Octopine dehydrogenase has been identified in the Mytilidae family for the first time, having previously only been established in one other marine invertebrate (P. maximus).

New insights into the systematics of North Atlantic Gaidropsarus (Gadiformes, Gadidae): flagging synonymies and hidden diversity

ABSTRACT Gaidropsarus Rafinesque, 1810 is a genus of marine fishes, commonly known as rocklings, comprising 14 living species and showing a high ecological diversity from the intertidal zone to the deep sea. The systematics of this group has been controversial due to a general lack of representative specimens and the conservative morphology exhibited. A multidisciplinary approach combining the analysis of meristic data and the DNA barcode standard was applied in a species delimitation approach. Individuals representing eight valid and three unnamed species were collected, morphologically identified and archived in several museum collections. Comparison of DNA sequences shows complex results, furthering the idea of the difficult identification of specimens based on traditional taxonomy. DNA barcoding supports synonymies, like G. biscayensis–G. macrophthalmus and G. guttatus–G. mediterraneus, agreeing with the extensive overlaps observed in the meristic variables analysed and suggesting a reduction in the number of species. Genetic distances showed pairs of closely related species like G. granti–G. vulgaris and G. argentatus–G. ensis, the latter being only distinguished by one main distinctive character. Four deep-water specimens, morphologically classified only to the genus level, constituted three independent taxa apart from the ones present in this study and with no barcode matches in the repository databases. They could represent new records for the North Atlantic or unknown species of this genus. The results obtained show that more studies will be necessary to solve the systematics of this branch of the Gadiformes.