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The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated with shared fisheries management. In an effort to resolve the species' stock dynamics, a genomewide search for spatially informative single nucleotide polymorphisms (SNPs) was undertaken, by way of sequencing reduced representation libraries. An allele frequency approach to SNP discovery was used, combining the data of 555 larvae and young-of-the-year (LYOY) into pools representing major geographical areas and mapping against a newly assembled genomic reference. From a set of 184,895 candidate loci, 384 were selected for validation using 167 LYOY. A highly discriminatory genotyping panel of 95 SNPs was ultimately developed by selecting loci with the most pronounced differences between western Atlantic and Mediterranean Sea LYOY. The panel was evaluated by genotyping a different set of LYOY (n = 326), and from these, 77.8% and 82.1% were correctly assigned to western Atlantic and Mediterranean Sea origins, respectively. The panel revealed temporally persistent differentiation among LYOY from the western Atlantic and Mediterranean Sea (FST = 0.008, p = .034). The composition of six mixed feeding aggregations in the Atlantic Ocean and Mediterranean Sea was characterized using genotypes from medium (n = 184) and large (n = 48) adults, applying population assignment and mixture analyses. The results provide evidence of persistent population structuring across broad geographic areas and extensive mixing in the Atlantic Ocean, particularly in the mid-Atlantic Bight and Gulf of St. Lawrence. The genomic reference and genotyping tools presented here constitute novel resources useful for future research and conservation efforts., This work was carried out under the provision of the ICCAT Atlantic-wide Research Programme for Bluefin Tuna (GBYP), funded by the European Union, by several ICCAT CPCs, the ICCAT Secretariat and other entities (see: http://www.iccat.int/GBYP/en/Budget.htm). Additional funds were provided by the MARES Joint Doctorate Programme Selected Under Erasmus Mundus and coordinated by Ghent University (FPA 2011-0016). Larvae from the Balearic Sea were provided by the ATAME project “Bluefin tuna abundance indices: towards recruitment estimators based on larval ecology” (CTM 2011-29525-C04-02) and the BLUEFIN project “Modelling bluefin spawning areas variability and population dynamics in the Western Mediterranean” (IEO-SOCIB agreement).

BackgroundThe blue shark (Prionace glauca, Linnaeus 1758) is one of the most abundant epipelagic shark inhabiting all the oceans except the poles, including the Mediterranean Sea, but its genetic structure has not been confirmed at basin and interoceanic distances. Past tagging programs in the Atlantic Ocean failed to find evidence of migration of blue sharks between the Mediterranean and the adjacent Atlantic, despite the extreme vagility of the species. Although the high rate of by-catch in the Mediterranean basin, to date no genetic study on Mediterranean blue shark was carried out, which constitutes a significant knowledge gap, considering that this population is classified as “Critically Endangered”, unlike its open-ocean counterpart.MethodsBlue shark phylogeography and demography in the Mediterranean Sea and North-Eastern Atlantic Ocean were inferred using two mitochondrial genes (Cytb and control region) amplified from 207 and 170 individuals respectively, collected from six localities across the Mediterranean and two from the North-Eastern Atlantic.ResultsAlthough no obvious pattern of geographical differentiation was apparent from the haplotype network, Φst analyses indicated significant genetic structure among four geographical groups. Demographic analyses suggest that these populations have experienced a constant population expansion in the last 0.4–0.1 million of years.DiscussionThe weak, but significant, differences in Mediterranean and adjacent North-eastern Atlantic blue sharks revealed a complex phylogeographic structure, which appears to reject the assumption of panmixia across the study area, but also supports a certain degree of population connectivity across the Strait of Gibraltar, despite the lack of evidence of migratory movements observed by tagging data. Analyses of spatial genetic structure in relation to sex-ratio and size could indicate some level of sex/stage biased migratory behaviour.

International audience; Global population genetic structure of yellowfin tuna (Thunnus albacares) is still poorly understood despite its relevance for the tuna fishery industry. Low levels of genetic differentiation among oceans speak in favour of the existence of a single panmictic population worldwide of this highly migratory fish. However, recent studies indicated genetic structuring at a much smaller geographic scales than previously considered, pointing out that YFT population genetic structure has not been properly assessed so far. In this study, we demonstrated for the first time, the utility of 2b-RAD genotyping technique for investigating population genetic diversity and differentiation in high gene-flow species. Running de novo pipeline in Stacks, a total of 6772 high-quality genome-wide SNPs were identified across Atlantic, Indian and Pacific population samples representing all major distribution areas. Preliminary analyses showed shallow but significant population structure among oceans (FST=0.0273; P-value

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions., peer-reviewed

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commer- cially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

The performance of different molecular markers in the assessment of population structure was tested using samples of Solea vulgaris collected in the Mediterranean within and outside the hypothetical dispersal ability of the species. A total of 172 individuals belonging to four population samples were analysed using 15 microsatellites [simple sequence repeats (SSRs)] and 153 amplified fragment length polymorphisms (AFLPs). Considering the global qualitative patterns, we found a correlation between SSRs and AFLPs in detecting genetic differentiation among samples. However, on a small geographical scale, AFLPs were able to discriminate individuals from neighbouring populations whereas SSRs were not, and the percentage of individuals correctly assigned to their population of origin was higher with AFLPs than with SSRs. The high number of loci analysed with the AFLP technique could increase the probability to include outlier loci in the analysis; however, the neutrality test performed on our data set did not show evidence of selection acting on the S. vulgaris samples. Even if the choice of the molecular marker depends mainly on the biological question to be addressed, the higher power of discrimination and the comparative technical ease of obtaining data from AFLPs with respect to SSRs suggest the use of AFLPs for many population genetics studies.

We used silver nitrate staining, CMA3 and FISH to study the chromosomal localization of both the major ribosomal genes and the nucleolar organizer regions as well as that of the minor ribosomal genes (5S rDNA) in two species of Batoidea, Taeniura lymma (Dasyatidae) and Raja montagui (Rajidae). In both species, all the metaphases examined showed the presence of multiple NOR-bearing sites, while the gene for 5S rRNA proved to be localized on two chromosome pairs. Furthermore, one of the two 5S rDNA sites in T. lymma was shown to be co-localized with the major ribosomal cluster. The presence of multiple nucleolar organizer regions in the two species might be interpreted as being the result of intraspecific polymorphisms, or as a phenomenon of the amplified transposition of mobile elements of the genome. We also determined the nucleotide sequence of the 5S rRNA gene, consisting of 564 bp in R. montagui and 612 bp in T. lymma. We also found TATA-like and (TGC) n trinucleotides, (CA) n dinucleotides and (GTGA) n tetranucleotides, which probably influence gene regulation.

In order to study the genetic structure of the Adriatic shared stock of red mullet (Mullus barbatus), we developed a set of dinucleotide microsatellite markers. A dinucleotide-enriched genomic library was obtained, and 6 polymorphic dinucleotide loci were successfully optimized. The markers showed high expected heterozygosity (from 0.68 to 0.92) and allele number (from 12 to 33); thus they appear to be suitable for detecting genetic differences in the population of red mullet. Four Adriatic samples were subsequently analyzed for microsatellite variation, and the results showed subtle but statistically significant genetic differentiation, indicating that the Adriatic red mullet may group into local, genetically isolated populations. No correlation between geographic distance and genetic differentiation was observed. In addition, the evidence of recent bottlenecks in the Adriatic samples indicates that the observed population subdivision might reflect random local allelic variations, generated by reproductive success, survival rates, or fishing pressure.

In the semienclosed Adriatic Sea, the shared stocks of the cephalopods Loligo vulgaris and Sepia officinalis represent important marine fisheries resources exploited by all coastal countries. The improving of knowledge on the demographic features of these shared stocks is internationally relevant for adopting responsible management and conservation of these marine resources. Analyses of microsatellite variation in geographical samples collected from all parts of the Adriatic Sea were performed using arrays of species-specific di-nucleotide and tri-nucleotide loci. In L. vulgaris the level of genetic variability was consistent with that observed in other loliginid species, whereas the S. officinalis stock showed a microsatellite variation markedly lower than that estimated for the Atlantic and Mediterranean populations collected around the Iberian peninsula. The weak spatial genetic differentiation, the discordant results of the genetic divergence estimators and the lack of any geographical cline in the spatial genetic differences suggest the occurrence of single genetically homogeneous populations within the Adriatic stocks of both species, recommending a coordinated management of the squid and cuttlefish by the Adriatic fishing countries. On the contrary, significant differences detected in temporal replicates of S. officinalis might suggest that allelic frequency can change relating to reproductive behaviour.

The smooth-hounds represent a significant proportion of the elasmobranch catch in the Adriatic basin of the Mediterranean Sea, where the common (Mustelus mustelus) and blackspotted (Mustelus punctulatus) smooth-hounds co-occur. The 2 species share several morphological and morphometric characters that lead to frequent misidentification. In order to provide information useful for their species identification, we performed a morphological identification of several Mustelus specimens to select individuals unambiguously attributed to 1 of the 2 species, and assayed these with 3 new molecular tests. First, we developed and validated a mitochondrial DNA assay based on species-specific amplification of the cytochrome c oxidase subunit 1 (COI). Second, a fragment analysis of 15 microsatellites cross-amplified from several triakid species was performed to identify diagnostic loci. Finally, a length difference was identified in the internal transcribed spacer 2 (ITS2) region and a diagnostic test based on its amplification was established. All the samples classified morphologically as M. mustelus and M. punctulatus showed a species-specific profile using all the 3 molecular tests. In addition, cross-amplification of microsatellites allowed identification of 9 highly polymorphic loci that will be useful for the study of the mating system and population differentiation of the 2 species.

The Bag320 sequence family of satellite DNA (satDNA) has been found in some stick insect taxa: the bisexual Bacillus grandii, the related parthenogen B. atticus and their hybrids with B. rossius. However, under the same experimental conditions, the Bag320 sequences were not found in B. rossius. Bag320 sequences of the clonal hybrid B. whitei (= B. rossius/grandii grandii) intermingled with those of B. grandii in all plotted dendrograms. On the whole, satDNA features (restriction pattern, sequence variation, fluorescent in situ hybridization (FISH)), allozymes and karyology support a relatively recent origin of B. whitei. Our investigations on unisexual hybrids of Bacillus also suggested that their origin and clonal reproduction allow the occurrence of different sequence subsets of limited variability in isolated populations stemming from the hybridization focus.

A microsatellite-enriched genomic library was obtained from the common sole, Solea vulgaris , and seven polymorphic dinucleotide markers were successfully optimized. These markers showed levels of expected heterozygosity ranging from 0.55 to 0.83 and allele number ranging from eight to 12. Three of these markers were also found to successfully amplify in the closely related Solea senegalensis and can be employed to define population genetic structure of the two Solea species and for inferring stock origins.

The Bag320 satellite DNA (satDNA) family was studied in seven populations of the stick insects Bacillus atticus (parthenogenetic, unisexual) and Bacillus grandii (bisexual). It was characterized as widespread in all zymoraces of B. atticus and in all subspecies of B. grandii. The copy number of this satellite is higher in the bisexual B. grandii (15%-20% of the genome) than in the parthenogenetic B. atticus (2%- 5% of the genome). The nucleotide sequences of 12 Bag320 clones from B. atticus and 17 from B. grandii differed at 13 characteristic positions by fixed nucleotide substitutions. Thus, nucleotide sequences from both species cluster conspecifically in phylogenetic dendrograms. The nucleotide sequences derived from B. grandii grandii could he clearly discriminated from those of B. grandii benazzii and B. grandii maretimi on the basis of 25 variable sites, although all taxa come from Sicily. In contrast, the Bag320 sequences from B. atticus could not he discriminated accordingly, although they derive from geographically quite distant populations of its three zymoraces (the Italian and Greek B. atticus atticus, the Greek and Turkish B. atticus carius, and the Cyprian B. atticus cyprius). The different rate of evolutionary turnover of the Bag320 satDNA in both species can he related to their different modes of reproduction. This indicates that meiosis and chromosome segregation affect processes in satDNA diversification.

Interspecific hybrids raise a variety of developmental, reproductive, and evolutionary issues. In Sicily, geographically and chronologically distinct hybridizations between the highly differentiated Bacillus rossius and B. grandii have produced hybridogenetic strains and clonal parthenogenetic species. In northern Sicily, all-female populations of facultatively Parthenogenetic B. rossius and bisexual B. grandii benazzii co-occur and we could test their current hybridization through electrophoretic marker analyses; control crosses with allopatric males were also carried out. Hybrid female progeny percentages ranged from 0 to 74 being fewer in egg batches laid by parthenogenetic mothers than in those of amphimictic females; no difference was noticed between sympatric and allopatric pairs. F1 hybrids of both sexes proved sterile; although some eggs started cleaving, no hemiclonal or clonal progeny hatched, only rare androgenetics being obtained. In currently produced hybrids a complete disruption of gametogenesis occurs, so that genetic constraints between parental taxa appear stronger now than in the past, most likely the result of ancestor evolution.

Populations of unisexual organisms are often assumed to be genetically invariant (clones) and destined to a short existence on an evolutionary timescale. Unisexual organisms are most often obligate parthenogens and, by definition, ought to be completely isolated reproductively from related bisexual organisms. The assumption of complete reproductive isolation between amphimictic ancestors and thelytokous hybrids is common to most hypotheses on the evolution of sex and its adaptive significance. Stick insects of the genus Bacillus however provide evidence for reproductive interactions between allodiploid parthenogens and their ancestors, because pure species progeny (an- drogenetics) and triploid descendants are produced. These findings demonstrate that, through androgenesis, offspring of parthenogenetic hybrid females can contribute specimens of both sexes to the fathering species when fertilized by syntopic ancestral males and the parthenogenetic egg of strictly clonal females, when fertilized, allows a third genome to be added to the allodiploid chromosome set. These triploid genomes promote further genetic diversification and evolution of the unisexual populations through the formation of new clones by recombination during the changed maturation mode of allotriploid eggs. All this argues for much more complex breeding systems and evolutionary pathways than are usually assumed for hybrid unisexual organisms.

Bacillus hybridogens represent the first example of hemiclonal reproduction among invertebrates and they were analyzed in the present study for comparison with vertebrate hybridogenetic complexes (Poeciliopsis and Rana). Experimental crosses of southeastern hybridogenetic B. rossius-grandii grandii females with both the parental species and hybrid males were carried out to test the reproductive potential of hybridogens. Allozyme and chromosome markers indicated that hemiclonal females actually reproduced in a variety of ways. Hybridogenesis was the most common and the rossius unassorted haploset was passed to the progeny while the grandii haploset was discarded during meiosis and replaced by that of the fathering male. Andro-genesis, which was much rarer, led to the production of two types of all-paternal offspring: unipa-rental progeny of both sexes and all-male progeny with the same allozyme and chromosome structure as their father. Mosaics of two kinds were also produced: the “h + h” type, with two different hybridogenetic cell lines; and the “h + a” type, with one hybridogenetic and one androgenetic cell line. Mosaics evolved into intersexes when their cell lines were sexually discordant. Among the evolutionary outcomes of this wide array of reproductive ways seems to be a shift from hemiclonality to clonality. This possibility is supported by the well-established parthenogen B. whitei and the occasional production of rare invariant specimens from the northwestern hybridogenetic form B. rossius-grandii benazzii. Bacillus and other unisexuals provide evidence of numerous reproductive interactions between hybrids and their bisexual relatives: the occurrence of such interactions may play an important role in the persistence, diversification and evolution of unisexuals. © 1995 Wiley-Liss, Inc.

The increasing number of recognized hybrid unisexual complexes among invertebrate and vertebrate animals has promoted investigations about their composition and origin. Morphological, karyological and genetic (protein and DNA) analyses clearly show that, owing to their persistence and incomplete reproductive isolation from ancestors, several all‐female complexes are much more diversified than generally assumed and that they may also have an evolutionary role. Here the case of the stick‐insects of the genus Bacillus is reported in some detail. This holomediterranean genus comprises three well differentiated species that in Sicily have hybridized repeatedly. The Bacillus mate‐recognition system has not followed the species‐specific differentiation of the allozyme‐coding loci, allowing interspecific crosses to occur in areas of species sym‐patry with the production of two hybridogens, a corresponding allodiploid parthenogen and a trihybrid triploid parthenogenetic species. Hybridogenetic females eli...

Details of six highly polymorphic dinucleotide and one highly polymorphic tetranucleotide microsatellite markers are provided for Pagellus erythrinus. These markers are highly polymorphic, with an expected heterozygosity ranging from 0.713 to 0.959 and allele numbers ranging from seven to 36. These microsatellite markers should help determine population genetic structure and fisheries stocks for management purposes.

A microsatellite-enriched genomic library was obtained from black-bellied angler Lophius budegassa and six polymorphic dinucleotide markers were successfully optimized. These markers showed levels of polymorphism ranging from 0.363 to 0.662 and allele numbers ranging from three to seven. These markers were also found to successfully amplify in the closely related Lophius piscatorius (angler) and thus appear to be useful for genetic stock structure analysis in both species.

The stick insects Bacillus rossius-grandii benazzii and B. rossius-grandii grandii naturally reproduce by hybridogenesis and androgenesis. The hybrid karyotype of the former (2n=35, XX female; 34, X0 male) clearly sums up a B. rossius haploset (r) with n=18 and a B. grandii benazzii one (gb) with n=17. The two sets keep the parental features for C-heterochromatin amount (much larger in the gb complement) and satellites/NORs (nuclear organizer regions) (more numerous and variably located in the r set); hybridogenetically produced males always show severely impaired gametogenesis and are therefore sterile, whereas hybridogenetic females are fertile. Reproductive, karyological and cytogenetical properties of the hybridogenetic system have been exploited to obtain the chromosomal evidence of whole haploset exchanges. In progenies obtained by crossing B. rossiusgrandii benazzii females to B. rossius males with either standard or repatterned (with Robertsonian fusions) karyotypes, there has always been complete agreement between electrophoretically genotyped and karyologically analyzed hybridogenetic offspring: the unassorted maternal r haploset (r m) is transmitted and the gb m haploset replaced by that of the fathering male (r p), thus evidencing the hemiclonal reproduction and the new r m-r p chromosomal constitution. New karyotype traits of the offspring relate to chromosome number (2n=36, female; 35, male), C-heterochromatin pattern (the heterochromatin-rich gb haploset completely disappears) and satellite/NOR features (corresponding to r m plus r p locations). The same crosses also produce genetically and chromosomally all-paternal descendants (androgenetics), of both sexes and fully fertile, with an r p r p structure. These androgenetic progeny show segregation for alleles and chromosomes at which fathering males are heterozygous: it was therefore possible to demonstrate that androgenetics can derive from syngamy of two sperm nuclei, of the several present in the polyspermic hybridogenetic egg. The production of androgenetics from field fertilized females of B. rossius-grandii benazzii, B. rossius-grandii grandii and parthenogenetic Bacillus whitei (=B. rossius/grandii grandii) suggests the occurrence of unsuspected relationships between hybrids and their parental species, so that the hybrids cannot be simply considered as ‘sexual parasites’. Furthermore, there is a suggestion of evolution of parthenogenetic clonal species from selection of initially hybridogenetic strains. The ability to produce uniparental progeny naturally from the spermatic genome may open a new field of investigation on genomic imprinting.

Background: International 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 Findings: This 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 hybridization experiments (, SCOPUS: ar.j, info:eu-repo/semantics/published

All demcs of Sardinian Bacillus atticus share a 2n = 33 karyotype which is fully comparable to the standard one (2n = 34), except for a Robertsonian fusion of the two chromosomes of pair 11. Furthermore, the San Vito population shows a cytotype with 34 elements, derived from the common 33‐chromosomes with the addition of an unusual, all‐heterochromatic large acrocentric one. C‐banding and silver staining have revealed a rather constant heterocromatin and NOR localizations in all samples; this suggests a most likely derivation of all Sardinian demes from common founder(s). The cytogenetic degree of differentiation of Sardinian B. atticus from other conspecific Italian demes, and from the bisexual B. grandii, is discussed. No cytogenetic evidence supporting the repeatedly stated hybrid structure of B. atticus is revealed from karyological studies, and the hybrid origin of B. atticus can be maintained on genetical grounds only. It is also suggested that the odd chromosome in the San Vito cytotype de...

Although considerable progress has been made in elucidating the relationships within the Chondrichthyes, there is no agreement as it concerns the systematics of Batoidea, the most derived superorder among cartilaginous fishes, and many different interpretations exist. Our investigation provides the first assessment of relationships among the described batoid species using sequences from both mtDNA and nuclear genes as well as karyological morphology. Our work consists primarily in reconstructing the phylogenetic relationships of Batoidea by examining the mtDNA (16S) and nuclear gene (18S) sequences from 11 batoid species. The three analytical methods (NJ, MP and Bayesian analysis) grouped Rajiformes, Myliobatiformes and Rhinobatiformes. In these trees the two torpedoes diverge from the other batoid fishes. We also compare the molecular data with the available karyological evidence, which consist of the diploid number and the karyotype morphology of eight species belonging to the four orders examined. The results show that the karyological structure in the different species is generally consistent with the various phylogenetical trees, and that Torpediniformes confirm their unique genome organization. © 2006 Elsevier B.V. All rights reserved.

The genomic and gene organisation of 5S rDNA clusters have been extensively characterized in bony fish and eukaryotes, providing general issues for understanding the molecular evolution of this multigene DNA family. By contrast, the 5S rDNA features have been rarely investigated in cartilaginous fish (only three species). Here, we provide evidence for a dual 5S rDNA gene system in the Rajidae by sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) in five Mediterranean species of rays (Rajidae), and in a large number of piscine taxa including lampreys and bony fish. As documented in several bony fish, two functional 5S rDNA types were found here also in the rajid genome: a short one (I) and a long one (II), distinguished by distinct 5S and NTS sequences. That the ancestral piscine genome had these two 5S rDNA loci might be argued from the occurrence of homologous dual gene systems that exist in several fish taxa and from 5S phylogenetic relationships. An extensive analysis of NTS-II sequences of Rajidae and Dasyatidae revealed the occurrence of large simple sequence repeat (SSR) regions that are formed by microsatellite arrays. The localization and organization of SSR within the NTS-II are conserved in Rajiformes since the Upper Cretaceous. The direct correlation between the SSRs extension and the NTS length indicated that they might play a role in the maintenance of the larger 5S rDNA clusters in rays. The phylogenetic analysis indicated that NTS-II is a valuable systematic tool limited to distantly related taxa of Rajiformes. © Springer Science+Business Media, Inc. 2006.

Heat shock proteins are a multigene family of polypeptides composed of the constitutively-expressed heat shock cognate (HSC) members and of the stress-inducible (HSP) proteins, whose expression is specifically induced by stress factors. Constitutive and inducible 70 kDa isoforms are reported in vertebrates and invertebrates. HSCs are expressed in all bivalve molluscs studied to date, while the occurrence of strictly heat-inducible HSPs seems a distinctive feature of oysters. To gain more insight into the molecular features of the Ostrea edulis HSP70, we have cloned and sequenced the gene product putatively encoding for the heat-inducible isoform HSP69 and examined the pattern of expression after heat exposure. Four different clones of approximately 1794 bp were obtained that share a high degree of homology with heat-inducible HSP70 from other bivalves. Amino acid sequence comparisons indicated that the main structural features of the heat-inducible HSP70 are highly conserved within the O. edulis HSP70 clones, while lower sequence homology occurred with respect to HSC70 transcripts. Northern blot analysis indicated that HSP69 mRNA was absent in control animals but induced after heat shock (1 h at 32 degrees C or higher). Induction was detectable immediately after heat shock, reaching a maximum after 2 to 3 h of post-stress recovery at 18 degrees C, and decreasing thereafter. A phylogenetic analysis of the HSP70 family members from oyster and other bivalves revealed a substantial conservation in the evolutionary pattern among constitutive and inducible gene products, from invertebrates to higher vertebrates.

A microsatellite dinucleotide-enriched library was obtained from the European squid (Loligo vulgaris) and five species-specific dinucleotide markers were optimized. These markers are highly polymorphic with average expected heterozygosity ranging from 0.706 to 0.927 and allele number ranging from 7 to 17. This set of primers is suitable for population genetic studies.