Andrés Sanjuan

Extensive population subdivision of the cuttlefish Sepia officinalis (Mollusca: Cephalopoda) around the Iberian Peninsula indicated by microsatellite DNA variation

The Atlantic Ocean-Mediterranean Sea junction has been proposed as an important phylogeographical area on the basis of concordance in genetic patterns observed at allozyme, mtDNA and microsatellite DNA markers in several marine species. This study presents microsatellite DNA data for a mobile invertebrate species in this area, the cuttlefish Sepia officinalis, allowing comparison of this relatively new class of DNA marker with previous allozyme results, and examination of the relative effects on gene flow of the Strait of Gibraltar and the Almería-Oran oceanographic front. Genetic variation at seven microsatellite loci screened in six samples from NE Atlantic and Mediterranean coasts of the Iberian Peninsula was high (mean Na = 9.6, mean He = 0.725). Microsatellites detected highly significant subpopulation structuring (FST= 0.061; RST = 0.104), consistent with an isolation-by-distance model of low levels of gene flow. Distinct and significant clinal changes in allele frequencies between Atlantic and Mediterranean samples found at five out of seven loci, however indicate these results might be also consistent with an alternative model of secondary contact and introgression between previously isolated and divergent populations, as previously proposed for other marine species from the Atlantic-Mediterranean area. A pronounced ‘step’ change between SW Mediterranean samples associated with the Almería-Oran front suggests this oceanographic feature may represent a contemporary barrier to gene flow.

A Microsatellite Genetic Map of the Turbot (Scophthalmus maximus)

A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 ± 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, ∼1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81–100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species.

Allozyme differentiation in the cuttlefish Sepia officinalis (Mollusca: Cephalopoda) from the NE Atlantic and Mediterranean

The Atlantic–Mediterranean area has recently been proposed as a new phylogeographical area on the basis of concordance of genetic differentiation patterns observed in several marine species. However, additional taxa need to be studied to establish the phylogeographical relationship between the Atlantic and Mediterranean. Eleven samples of the cuttlefish Sepia officinalis around the Iberian Peninsula, one from the Canary Islands, and another from Fiumicino (Italy) were screened for 33 allozyme loci. Genetic variability was low in all samples (He between 0.022 and 0.076). Intersample genetic differentiation was high (FST=0.220), mainly because of genetic variation in the non-Iberian samples. One locus (PEPD*), diagnostic between the Italian sample and all others, suggests the possible existence of hitherto unrecognized species or subspecies of Sepia in the Mediterranean Sea. The 11 Iberian samples exhibited moderate genetic differentiation (FST=0.100), which could be explained on the basis of genetic differentiation between Atlantic and Mediterranean samples. Significant clines in allele frequencies were observed for five out of six polymorphic loci. These results support a model of secondary intergradation (i.e. secondary contact of populations that were previously differentiated in isolation) similar to that previously proposed for other marine species from the Atlantic–Mediterranean area.

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.

Genetic differentiation in three Sepia species (Mollusca: Cephalopoda) from Galician waters (north-west Iberian Peninsula)

The systematics of the genus Sepia is not yet clear. Morphological evidence has led to S. officinalis Linnaeus, 1758 being considered as belonging to the subgenus Sepia sensu stricto, and S. orbignyana Férussac, 1826 and S. elegans Blainville, 1827 as belonging to the subgenus Rhombosepion. Samples of 30 individuals of S. officinalis and S. orbignyana from both sides of an oceanographic boundary off the north-west Iberian Peninsula, and a sample of S. elegans from the northern side, were collected in 1993–1994. Allozyme electrophoresis for 32 presumptive loci revealed low levels of genetic variability for the three Sepia species (mean expected heterozygosity estimates were <0.052). No significant differences in allozyme frequencies were detected among populations of either S. officinalis or S. orbignyana. The genetic identities (I) of S. officinalis and S. orbignyana (I=0.12) and of S. elegans (I=0.13) were significantly different from that of S. orbignyana and S. elegans (I=0.49). The former are typical of values for confamilial genera, and a new generic status is proposed for the latter two species, which become Rhombosepion orbignyana (Férussac, 1826) and R. elegans (Blainville, 1827).

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.

Species differentiation of Sepia officinalis and Sepia hierredda (Cephalopoda: Sepiidae) based on morphological and allozyme analyses

Two taxa of commercially exploited cuttlefish, Sepia officinalis and S. hierredda , are compared for the first time on the bases of quantitative morphology and allozyme polymorphisms. Morphometric measurements and meristic counts of selected soft and hard (cuttlebone) body characters, with allozyme electrophoretic analysis are used. Samples were obtained from north-west Iberian Peninsula and Senegalese waters (West Africa). Significant differences in mantle width, arm and hectocotylus length, numbers of rows of reduced suckers on the hectocotylus and in most cuttlebone measurements were found. Canonical discriminant functions of cuttlebone measurements for males and females were calculated. Allozyme electrophoresis for 33 presumptive loci showed low levels of genetic variability and 13 diagnostic loci between the two Sepia taxa. The genetic identities ( I ) in pairwise comparisons of populations of both taxa were I =0·582–0·596, which are typical values for congeneric species. These congruent morphological and genetic results strongly suggest that S. officinalis and S. hierredda are different species.

Allozyme electrophoretic technique and phylogenetic relationships in three species of Sepia (Cephalopoda: Sepiidae) ☆

Samples of Sepia officinalis, S. orbignyana, and S. elegans from Galician waters (NW Iberian Peninsula) were obtained from commercial catches in three fishing ports. A preliminary screening for 47 and 33 enzymes in mantle muscle and digestive gland, respectively, was carried out using seven buffer systems. Thirty-seven enzyme loci were resolved from these tissues using only two buffer systems. Most enzymes showed equal or higher activity for the digestive gland than for the mantle muscle in freshly-caught samples of S. officinalis and S. orbignyana. The activity of a large number of enzymes decreased faster in the digestive gland than in the mantle muscle after 6–12 h at room temperature. Consequently, we suggest that mantle muscle rather than digestive gland be used for routine electrophoretic studies in Sepia species obtained from commercial catches. A phylogenetic reconstruction analysis, applying the Wagner parsimony method and using the ommastrephid species Illex coindetii as outgroup, showed only one most parsimonious tree. S. orbignyana and S. elegans were found to be a sister group. The topology agreed with that recently obtained from mitochondrial rDNA sequences, and both molecular data are in line with previous morphological results. They confirm the view that S. orbignyana and S. elegans belong to a different subgenus (Rhombosepion) from that of S. officinalis (Sepia “sensu stricto”).