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[Resumen] El camarón Palaemon elegans es un crustáceo decápodo distribuido a lo largo de toda la costa europea y caracterizado por su capacidad de adaptación a condiciones ambientales extremadamente variables. El amplio nicho ecológico y la reciente expansión geográfica de esta especie determinan que sea considerada como una especie importante dentro de la fauna litoral europea. En este trabajo se estudia la estructura y la diversidad genética de las poblaciones de P. elegans empleando marcadores moleculares mitocondriales y nucleares. Los resultados obtenidos revelan la compleja filogeografía de esta especie, determinada por su historia evolutiva y la existencia actual de barreras al flujo génico entre las diferentes poblaciones. Asimismo, se detectó una elevada diversidad genética en todas las localidades analizadas, con la excepción de aquellas en las que esta especie ha sido introducida por acción humana y en las que ha adquirido un carácter invasor. Finalmente, se desarrollaron y caracterizaron marcadores microsatélite por primera vez en esta especie de camarón, proporcionándose así nuevas herramientas moleculares que permitirán profundizar en la genética de poblaciones de esta especie, entre otras posibles aplicaciones de interés., [Abstract] Shrimp Palaemon elegans is a decapod crustacean distributed along the entire European coast. This species is characterized by its ability to adapt to highly variable enviromental conditions. Due to its broad ecological niche and recent geographic expansion, this shrimp species is considered an important species in European coastal fauna. Genetic structure and genetic diversity of P. elegans populations were studied in this work using mitochondrial and nuclear molecular markers. The obtained results show a complex phylogeography in P. elegans, determined by its evolutionary history and the current existence of barriers to gene flow between different populations. High genetic diversity was detected in most of the analyzed locations. Locations where P. elegans has been introduced by human action show low genetic diversity, acquiring this species an invasive behavior. Finally, microsatellite markers were developed and characterized for the first time in this shrimp species. New molecular tools, provided by this work, will allow to go into detail about population genetics of this species, among other possible applications of interest.

This study documents the introduction of the European Baltic prawn, Palaemon adspersus Rathke, 1837 to the coastal waters of northeastern North America, specifically the west coast of Newfoundland and the Magdalen Islands in the Gulf of St. Lawrence, Canada. Species identification was verified using morphological and genetic criteria. In September 2011, the first specimens of P. adspersus were collected in Gulf of St. Lawrence waters near Stephenville Crossing, Newfoundland, Canada. In 2012, additional P. adspersus specimens were collected in this area and at St. Andrew’s, located further south in western Newfoundland and in 2013 several egg-bearing females were collected further north in York Harbour. Accidental transport by ballast water of ships seems the likely vector for transport of Baltic prawn to the Gulf of St. Lawrence from Northern Europe or the Caspian Sea. It is possible that this shrimp has a wider presence in Atlantic Canadian waters but, due to its close resemblance to native shrimp species, it may have been previously misidentified, as occurred with specimens collected from the Magdalen Islands. We further expect that other species of the genus Palaemon, including P. elegans Rathke, 1837 from the Baltic Sea or northeastern United States, and P. macrodactylus Rathbun, 1902 from the northeastern United States, may invade the Gulf of St. Lawrence. We provide an illustrated key for the identification of these exotic Palaemon species and to differentiate them from native members of the subfamily Palaemoninae.

[Resumen] En esta tesis se ha realizado un estudio genético de un grupo de bivalvos marinos de la familia Pharidae Adams y Adams, 1858, y principalmente de las navajas del género Ensis Schumacher, 1817. El estudio se desarrolla dentro de las áreas de la genética evolutiva, genética poblacional, citogenética, delimitación de especies y DNA barcoding. Las especies de navaja utilizadas fueron seleccionadas debido al interés comercial que muchas especies de Ensis tienen en varias regiones de Europa y América, incluyéndose también el litoral de Galicia. Además, su distribución a ambos lados del Atlántico y en zonas de la costa pacífica de los Estados Unidos, El Perú y Chile hace que sean de interés en estudios filogeográficos. Por último, los problemas en la identificación de las especies a partir de caracteres morfológicos hicieron de este grupo de organismos un buen candidato para el desarrollo de herramientas moleculares y citogenéticas que complementasen los estudios de morfometría. La metodología utilizada varía en cierto modo a lo largo de cada uno de los capítulos que conforman los resultados de la tesis, aunque puede resumirse como sigue: en el capítulo 4.1.1, el ADN ribosomal 5S y el ADN pequeño nuclear U1, que conforman familias génicas de copia múltiple, fueron estudiados mediante un procedimiento de amplificación por PCR, clonación y secuenciación. Todas estas secuencias obtenidas experimentalmente en el laboratorio fueron posteriormente analizadas mediante numerosas herramientas bioinformáticas. En el capítulo 4.1.2, en el que se estudiaron 97 especies de metazoos, la metodología utilizada fue diferente, ya que se utilizaron bases de datos de proyectos genoma y herramientas bioinformáticas de alta capacidad, algunas de las cuales fueron diseñadas expresamente para este trabajo. En el capítulo 4.2 se realizó un estudio citogenético en el que se utilizaron técnicas de microscopía en campo claro y fluorescente, así como la hibridación de sondas fluorescentes so

[Abstract] In this study 4310 expressed sequence tags (ESTs) were used to identify potentially useful transcripts for future studies in the gooseneck barnacle Pollicipes pollicipes (Gmelin, 1789). 119 ESTs were obtained in this work and 4191 were taken from Meusemann et al. (2010). The gooseneck barnacle is a sessile pedunculate cirripede of great economic importance that occurs in dense aggregations, and is harvested for human consumption. The assembly of these ESTs yielded 1805 unigenes (461 contigs and 1344 singlets). The identification of cement proteins in our data is particularly interesting for cirripedes. Only a small part of the assembled unigenes could be functionally annotated. However, our results greatly improve our understanding of the biological features of P. pollicipes. In addition to this, a large number of potentially interesting genes were identified in order to serve as the base for future evolutionary studies in P. pollicipes.

[Abstract] Several studies on 5S ribosomal DNA (5S rDNA) have been focused on a subset of the following features in mostly one organism: number of copies, pseudogenes, secondary structure, promoter and terminator characteristics, genomic arrangements, types of non transcribed spacers and evolution. In this work, we systematically analyzed 5S rDNA sequence diversity in available metazoan genomes, and showed organism-specific and evolutionary-conserved features. Putatively functional sequences (12 766) from 97 organisms allowed us to identify general features of this multigene family in animals. Interestingly, we show that each mammal species has a highly conserved (housekeeping) 5S rRNA type and many variable ones. The genomic organization of 5S rDNA is still under debate. Here, we report the occurrence of several paralog 5S rRNA sequences in 58 of the examined species, and a flexible genome organization of 5S rDNA in animals. We found heterogeneous 5S rDNA clusters in several species, supporting the hypothesis of an exchange of 5S rDNA from one locus to another. A rather high degree of variation of upstream, internal and downstream putative regulatory regions appears to characterize metazoan 5S rDNA. We systematically studied the internal promoters and described three different types of termination signals, as well as variable distances between the coding region and the typical termination signal. Finally, we present a statistical method for detection of linkage among noncoding RNA (ncRNA) gene families. This method showed no evolutionary-conserved linkage among 5S rDNAs and any other ncRNA genes within Metazoa, even though we found 5S rDNA to be linked to various ncRNAs in several clades.

[Abstract] Ensis Schumacher, 1817 razor shells occur at both sides of the Atlantic and along the Pacific coasts of tropical west America, Peru, and Chile. Many of them are marketed in various regions. However, the absence of clear autapomorphies in the shell and the sympatric distributions of some species often prevent a correct identification of specimens. As a consequence, populations cannot be properly managed, and edible species are almost always mislabelled along the production chain. In this work, we studied whether the currently accepted Atlantic Ensis morphospecies are different evolutionary lineages, to clarify their taxonomic status and enable molecular identifications through DNA barcoding. For this, we studied 109 specimens sampled at 27 sites, which were identified as belonging to nine of those morphospecies. We analysed nucleotide variation at four nuclear (18S, 5.8S, ITS1, and ITS2) and two mitochondrial (COI and 16S) regions, although the 18S and 5.8S regions were not informative at the species level and were not further considered. The phylogenetic trees and networks obtained supported all morphospecies as separately evolving lineages. Phylogenetic trees recovered Ensis at each side of the Atlantic as reciprocally monophyletic. Remarkably, we confirm the co-occurrence of the morphologically similar E. minor (Chenu, 1843) and E. siliqua (Linné, 1758) along the NW Iberian coast, a fact that has been often overlooked. In South America, a relevant divergence between E. macha (Molina, 1792) individuals from Chile and Argentina was unveiled and suggests incipient speciation. We also confirm the occurrence of the North American species E. directus (Conrad, 1843) as far south as north-eastern Florida. Among the genomic regions analysed, we suggest COI as the most suitable DNA barcode for Atlantic Ensis. Our results will contribute to the conservation and management of Ensis populations and will enable reliable identifications of the edible species, even in

[Abstract] The European razor shell Ensis minor (Chenu 1843) and the American E. directus (Conrad 1843) have a diploid chromosome number of 38 and remarkable differences in their karyotypes: E. minor has four metacentric, one metacentric–submetacentric, five submetacentric, one subtelocentric and eight telocentric chromosome pairs, whereas E. directus has three metacentric, two metacentric–submetacentric, six submetacentric, six subtelocentric and two telocentric pairs. Fluorescent in situ hybridisation (FISH) using a major ribosomal DNA probe located the major ribosomal genes on one submetacentric chromosome pair in both species; FISH with a 5S ribosomal DNA (5S rDNA) probe rendered one chromosomal (weak) signal for E. minor and no signal for E. directus, supporting a more dispersed organisation of 5S rDNA compared to the major ribosomal genes. The vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome, and no interstitial signals were detected. In this work, a comparative karyological analysis was also performed between the four Ensis species analysed revealing that the three European species studied so far, namely E. minor, E. siliqua (Linné 1758) and E. magnus Schumacher 1817 show more similarities among them than compared to the American species E. directus. In addition, clear karyotype differences were found between the morphologically similar species E. minor and E. siliqua.

[Abstract] We report current genetic variation of populations of the razor shell Ensis directus (Conrad 1843) (Mollusca: Bivalvia: Pharidae) in native (North American) and introduced (European) ranges using nuclear and mitochondrial sequence-based markers. We expected less variation within the introduced range, especially considering the frequent mass mortality events observed in Europe since the species was recorded for the first time in 1978. However, we found higher variation in Europe. The possible significance of temporal fluctuations of genetic variation, limited effect of random genetic drift, and multiple introductions are discussed. Interestingly, the multiple-introduction hypothesis contrasts with the gradual colonisation of European coastal waters but is supported by trained clustering analysis and by the intensity of transatlantic shipping. Genetic and morphometric evidence strongly supports that examined individuals from a supposed E. directus population from Newfoundland (Canada) belong to a separate species. This new Ensis is formally described here and named E. terranovensis n.sp.

[Abstract] The linkage between 5S ribosomal DNA and other multigene families has been detected in many eukaryote lineages, but whether it provides any selective advantage remains unclear. In this work, we report the occurrence of linked units of 5S ribosomal DNA (5S rDNA) and U1 small nuclear DNA (U1 snDNA) in 10 razor shell species (Mollusca: Bivalvia: Pharidae) from four different genera. We obtained several clones containing partial or complete repeats of both multigene families in which both types of genes displayed the same orientation. We provide a comprehensive collection of razor shell 5S rDNA clones, both with linked and nonlinked organisation, and the first bivalve U1 snDNA sequences. We predicted the secondary structures and characterised the upstream and downstream conserved elements, including a region at −25 nucleotides from both 5S rDNA and U1 snDNA transcription start sites. The analysis of 5S rDNA showed that some nontranscribed spacers (NTSs) are more closely related to NTSs from other species (and genera) than to NTSs from the species they were retrieved from, suggesting birth-and-death evolution and ancestral polymorphism. Nucleotide conservation within the functional regions suggests the involvement of purifying selection, unequal crossing-overs and gene conversions. Taking into account this and other studies, we discuss the possible mechanisms by which both multigene families could have become linked in the Pharidae lineage. The reason why 5S rDNA is often found linked to other multigene families seems to be the result of stochastic processes within genomes in which its high copy number is determinant

[Abstract] Background: The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. Results: The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. Conclusions: These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.

[Abstract] The aim of this study was to analyse the genetic diversity and population structure in the razor clam Ensis siliqua along the European Atlantic coast taking into account their recent history of exploitation and the ‘Prestige' oil spill. To this end we examined the genetic variability of microsatellite markers in 211 razor clams from five populations in Ireland, Portugal and Spain. Microsatellite data revealed a low genetic differentiation between the Spanish and Portuguese populations (FST = 0–0.032) and a moderate differentiation of these populations and the Irish samples (FST = 0.071–0.100). Although we observed changes in genetic diversity in accordance with the level of exploitation and the distribution of the oil spill, these changes were mild and not significant after Bonferroni correction. This could be the result of a genuine low impact, lack of statistical power and/or the capacity of this species to recolonize quickly after the impact of anthropogenic stressors. Supporting the latter argument we found a significant temporal heterogeneity of allelic frequencies in samples coming from the same sampling locality that could be attributed to the movement of adults or larvae from unaffected source populations.

[Abstract] Internal transcribed spacer 1 and 2 (ITS1 and ITS2) sequences were analysed in Ensis razor shells (Mollusca: Bivalvia: Pharidae). We aimed to (1) test ITS1 and ITS2 as molecular markers at the population level in the successful alien E. directus (Conrad, 1843); (2) test these spacers at the species level in E. directus and three other Ensis species, E. siliqua (L., 1758), E. macha (Molina, 1782), and E. magnus (Schumacher, 1817); and (3) analyse the evolutionary processes that may be shaping Ensis ITS1 and ITS2 extant variation. In E. directus, despite the intragenomic divergence detected, ITS1 and ITS2 were informative in differentiating the geographic areas considered (Denmark and Canada) by means of both the insertion-deletion polymorphism and the nucleotide polymorphism. In this species, the 5.8S ribosomal gene (5.8S) showed scarce polymorphism. At the species level, maximum parsimony and maximum likelihood analyses revealed that ITS1 and ITS2 may be suitable to reconstruct Ensis phylogenetic relationships. Finally, the evolutionary models that best fit the long-term evolution of Ensis ITS1–5.8S–ITS2 are discussed. A mixed process of concerted evolution, birth-and-death evolution, and selection is chosen as an option that may reconcile the long-term evolution of Ensis ITS1–5.8S–ITS2 and 5S ribosomal DNA.

[Abstract] A study of nucleotide sequence variation of 5S ribosomal DNA from six Ensis species revealed that several 5S ribosomal DNA variants, based on differences in their nontranscribed spacers (NTS), occur in Ensis genomes. The 5S rRNA gene was not very polymorphic, compared with the NTS region. The phylogenetic analyses performed showed a between-species clustering of 5S ribosomal DNA variants. Sequence divergence levels between variants were very large, revealing a lack of sequence homogenization. These results strongly suggest that the long-term evolution of Ensis 5S ribosomal DNA is driven by birth-and-death processes and selection.

[Abstract] The aim of this study was to characterize new microsatellite loci in the razor clam Ensis arcuatus (Bivalvia: Pharidae) and examine the temporal genetic variability of a natural bed in Cies Islands (Galicia, north-western Spain) exploited by apnoea divers and affected by the ‘Prestige’ oil tanker spill in November 2002. In this work, we characterized four polymorphic microsatellites using an alternative approach that relies on the amplification and sequencing of ISSR markers. Observed heterozygosities ranged from 0.1053–0.6800 and number of alleles from 4–19. Linkage equilibrium was observed in all loci and two of them showed significant deviations from Hardy–Weinberg equilibrium. Estimators of FST between samples were low (<0.05) and not different from zero with a confidence level of 5%. We did not detect a clear decreasing tendency in genetic diversity although we found a significant change in allelic frequencies among samples (P = 0.0024) after the ‘Prestige’ oil spill. We propose that both phenomena could be related to a high variance in genetic success and/or a movement of adults or larvae from different source populations.

[Abstract] The razor clams Ensis arcuatus and E. siliqua show a diploid DNA content of 3.85 ± 0.049 pg and 4.00 ± 0.050 pg, respectively. Both have a diploid chromosome number of 38 although their karyotypes show remarkable differences. The karyotype of E. arcuatus consists of 4 metacentric, 1 metacentric-submetacentric, 7 submetacentric and 7 telocentric chromosome pairs, whereas that of E. siliqua possesses 3 metacentric, 7 submetacentric and 9 telocentric pairs. In situ hybridization using an 18S-5.8S-28S rDNA probe located this ribosomal locus on one chromosome pair for both species. Results demonstrate that large differences exist between them, probably caused by chromosome rearrangements along evolution of these two species, and increase the number of studies on bivalve cytogenetics.

[Abstract] Some aspects of microsatellite evolution, such as the role of base substitutions, are far from being fully understood. To examine the significance of base substitutions underlying the evolution of microsatellites we explored the nature and the distribution of interruptions in dinucleotide repeats from the human genome. The frequencies that we inferred in the repetitive sequences were statistically different from the frequencies observed in other noncoding sequences. Additionally, we detected that the interruptions tended to be towards the ends of the microsatellites and 5′-3′ asymmetry. In all the estimates nucleotides forming the same repetitive motif seem to be affected by different base substitution rates in AC and AG. This tendency itself could generate patterning and similarity in flanking sequences and reconcile these phenomena with the high mutation rate found in flanking sequences without invoking convergent evolution. Nevertheless, our data suggest that there is a regional bias in the substitution pattern of microsatellites. The accumulation of random substitutions alone cannot explain the heterogeneity and the asymmetry of interruptions found in this study or the relative frequency of different compound microsatellites in the human genome. Therefore, we cannot rule out the possibility of a mutational bias leading to convergent or parallel evolution in flanking sequences.

[Abstract] Four microsatellite loci in the razor clam Solen marginatus are described. Loci were isolated from the sequences of intersimple sequence repeat (ISSR) markers and an enriched library. Detailed analysis of these sequences led to the design of eight primer pairs. Allelic variation was assessed in 20 individuals from Redondela, Spain. The genetic variation observed in the markers presented here will be useful for future studies on the population structure of Solen marginatus in the wild and for aquaculture of this species.

[Abstract] Ensis siliqua is regarded as an increasingly valuable fishery resource with potential for commercial aquaculture in many European countries. The genetic variation of this razor clam was analysed by randomly amplified polymorphic DNA (RAPD) in six populations from Spain, Portugal and Ireland. Out of the 40 primers tested, five were chosen to assess genetic variation. A total of 61 RAPD loci were developed ranging in size from 400 to 2000 bp. The percentages of polymorphic loci, the allele effective number and the genetic diversity were comparable among populations, and demonstrated a high level of genetic variability. The values of Nei's genetic distance were small among the Spanish and Portuguese populations (0.051–0.065), and high between these and the Irish populations. Cluster and principal coordinate analyses supported these findings. A mantel test performed between geographic and genetic distance matrices showed a significant correlation (r=0.84, P<0.05), suggesting an isolation by distance process.

[Abstract] A phylogenetic reconstruction based on the amplification of 3 satellite DNAs (stDNAs) was carried out in 1 crustacean species and 15 bivalve species of the subclass Pteriomorphia (10, subfamily Mytilinae; 1, subfamily Litophaginae; 1, subfamily Modiolinae, all belonging to family Mytilidae; 1, family Arcidae; and 2, family Pectinidae). The sequences obtained showed motifs with high similarity to those of A and B boxes of tRNA promoter regions. Dot-blot hybridizations revealed that the 3 stDNAs are present mainly in high copy numbers for each species of the genus Mytilus, whereas for the other species they appear in low copy numbers. Maximum-parsimony trees evidenced a tendency to group Mytilus clones together, and species containing these sequences as a single copy were distributed among the different mytilids. Finally, the possible origin and evolution of these stDNAs is discussed.

[Abstract:] Histones are small basic nuclear proteins with critical structural and functional roles in eukaryotic genomes. The H1 multigene family constitutes a very interesting histone class gathering the greatest number of isoforms, with many different arrangements in the genome, including clustered and solitary genes, and showing replication-dependent (RD) or replication-independent (RI) expression patterns. The evolution of H1 histones has been classically explained by concerted evolution through a rapid process of interlocus recombination or gene conversion. Given such intriguing features, we have analyzed the long-term evolutionary pattern of the H1 multigene family through the evaluation of the relative importance of gene conversion, point mutation, and selection in generating and maintaining the different H1 subtypes. We have found the presence of an extensive silent nucleotide divergence, both within and between species, which is always significantly greater than the nonsilent variation, indicating that purifying selection is the major factor maintaining H1 protein homogeneity. The results obtained from phylogenetic analysis reveal that different H1 subtypes are no more closely related within than between species, as they cluster by type in the topologies, and that both RD and RI H1 variants follow the same evolutionary pattern. These findings suggest that H1 histones have not been subject to any significant effect of interlocus recombination or concerted evolution. However, the diversification of the H1 isoforms seems to be enhanced primarily by mutation and selection, where genes are subject to birth-and-death evolution with strong purifying selection at the protein level. This model is able to explain not only the generation and diversification of RD H1 isoforms but also the origin and long-term persistence of orphon RI H1 subtypes in the genome, something that is still unclear, assuming concerted evolution.

[Abstract] The present work represents the first characterization of a clustered histone repetitive unit containing an H1 gene in a bivalve mollusk. To complete the knowledge on the evolutionary history of the histone multigene family in invertebrates, we undertake its characterization in five mussel Mytilus species, as an extension of our previous work on the H1 gene family. We report the quintet H4–H2B–H2A–H3–H1 as the major organization unit in the genome of Mytilus galloprovincialis with two 5S rRNA genes with interspersed nontranscribed spacer segments linked to the unit, which is not justified by their cotranscription with histone genes. Surprisingly, 3′ UTR regions of histone genes show two different mRNA termination signals, a stem-loop and a polyadenylation signal, both related to the evolution of histone gene expression patterns throughout the cell cycle. The clustered H1 histones characterized share essential features with “orphon” H1 genes, suggesting a common evolutionary origin for both histone subtypes which is supported by the reconstructed phylogeny for H1 genes. The characterization of histone genes in four additional Mytilus species revealed the presence of strong purifying selection acting among the members of the family. The chromosomal location of most of the core histone genes studied was identified by FISH close to telomeric regions in M. galloprovincialis. Further analysis on nucleotide variation would be necessary to assess if H1 proteins evolve according to the birth-and-death model of evolution and if the effect of the strong purifying selection maintaining protein homogeneity could account for the homologies detected between clustered and “orphon” variants.

[Resumen] Se realiza un análisis poblacional, mediante marcadores RAPDs (Random Amplified Polymorphic DNA), de la estructura genética de 12 poblaciones de mejillón pertenecientes al género Mytilus, procedentes de diferentes lugares geográficos. Este trabajo constituye el primer ensayo, aplicando este tipo de marcador molecular, en un número tan elevado de poblaciones de mejillón. Los resultados obtenidos reflejan, en base al análisis de 32 loci polimórficos, la existencia de una elevada variabilidad genética, tanto intrapoblacional como intraespecífica y, como consecuencia, revelan el establecimiento de una estructura genética definida. Por otra parte, se ha detectado la existencia de una estructura panmíctica a nivel de las 3 localidades gallegas de M.galloprovincialis, así como un flujo genético restringido entre las poblaciones europeas de mejillón, probablemente relacionado con la existencia de barreras genéticas a lo largo de la costa Oeste de Europa. El conjunto de análisis realizados, que incluyen componentes princiaples, análisis de la varianza molecular, estimadores de distancia, índices de fijación y diferentes topologías, también reveló la posible presencia de individuos híbridos M.edulis-M.trossulus en la población de Isle of Man, así como la posible influencia humana en el establecimiento del flujo genético. El análisis de las especies japonesas que cohabitan la Bahía de Otsuchi (M.galloprovincialis y M.coruscus) reveló una mayor proximidad de éstas a M.californianus que a culaquier otra forma de mejillón. Asimismo, se han desarrollado marcadores, diagnóstico que permiten identificar inequívocamente determinadas especies de mejillón de interés comercial con el fin de evitar posibles fraudes, lo cual, desde el punto de vista de su aplicación industrial, resulta de un elevado interés. En este sentido se ha desarrollado un marcador que permite identificar stocks de poblaciones de M.galloprovincia

[Abstract]: The hexamer repeat sequence (TTAGGG) n, found at the ends of all vertebrate chromosomes, was previously identified as the main building element of one member of aHindIII satellite DNA family characterized in the genome of the bivalve mollusc "Donax trunculus". It was also found in 22 perfect tandem repeats in a cloned junction region juxtaposed to the proper satellite sequence, from which the DNA tract encompassing the clustered tandem copies was excised and subcloned. Here, the chromosomal distribution of (TTAGGG) n sequences in the "Donax" was studied by the sensitivity to Bal31 exonuclease digestion, fluorescence in situ hybridization (FISH) on metaphase chromosomes and rotating-field gel electrophoresis. To verify the occurrence of the hexamer repeat in the genomes of taxonomically related molluscs and other marine invertebrates, genomic DNA from the mussel "Mytilus galloprovincialis" and the echinoderm "Holothuria tubulosa" was also analyzed. The kinetics of Bal31 hydrolysis of high molecular mass DNA from the three marine invertebrates revealed a marked decrease over time of the hybridization with the cloned (TTAGGG)22 sequence, concomitantly with a progressive shortening of the positively reacting DNA fragments. This revealed a marked susceptibility to exonuclease consistent with terminal positioning on the respective chromosomal DNAs. In full agreement, FISH results with the (TTAGGG) 22 probe showed that the repeat appears located in telomeric regions in all chromosomes of both bivalve molluscs. The presence of (TTAGGG) n repeat tracts in marine invertebrate telomeres points to its wider distribution among eukaryotic organisms and suggests an ancestry older than originally presumed from its vertebrate distinctiveness.

[Abstract]: The chromosomes of “Donax trunculus” were analysed by means of Giemsa staining, chromomycin A3 (CA3), DAPI and fluorescent “in situ” hybridization (FISH) with an 18S-5.8S-28S rDNA probe. The diploid number is 38 chromosomes and the karyotype consists of nine pairs of metacentric chromosomes, two pairs of submetacentric-metacentric, seven pairs of submetacentric and one pair of telocentric chromosomes. CA3-positive bands are located on eight chromosome pairs and DAPI treatment resulted in uniform staining. Major ribosomal clusters 18S-5.8S-28S are located on the short arm of one submetacentric chromosome pair.

[Abstract:] Linker histones are a divergent group of histone proteins with an independent evolutionary history in which, besides somatic subtypes, tissue- and differentiation-specific subtypes are included. In the present work H1 histone coding and noncoding segments from five Mytilus mussel species (Mollusca: Bivalvia) widely distributed throughout the world have been determined and characterized. Analysis of promoter regions shows clear homologies among Mytilus H1 genes, sea urchin H1 genes, and vertebrate differentiation-specific H1 subtypes (H5 and H10), all having an H4 box motif in common. The amino acid sequence of the H1 protein central conserved domain is also closely related to that previously defined for the vertebrate divergent subtypes. A phylogenetic tree reconstructed from different H1 genes from several species strengthens the hypothesis of an “orphon” origin for the Mytilus H1 genes, as well as for the H10/H5 genes from vertebrates and the H1D gene from the sea urchin Strongylocentrotus purpuratus, is suggested. As additional data, the average copy number of the H1 genes in the species analyzed was estimated as being 100 to 110 copies per haploid genome, where FISH revealed telomeric chromosomal location for several H1 copies in M. galloprovincialis. The contribution of such proximity to heterochromatic regions over the amount of codon bias detected for H1 genes is discussed.

[Abstract:] We report the characterization of three satellite DNAs in four species of mussel: Mytilus edulis, Mytilus galloprovincialis, Mytilus trossulus, and Mytilus californianus. The monomers of the Apa I satellite DNAs were 173, 161, and 166 bp long. These satellite monomers were used to construct phylogenetic trees to infer relationships among these species. The topologies obtained clearly indicate that M. californianus is the most divergent species with respect to the other three. Furthermore, localization of satellite DNAs on metaphase chromosomes was performed using fluorescent in situ hybridization (FISH). Fluorescent signals revealed a different organization and distribution of these three satellite DNAs.

[Resumen] Este trabajo se realizó en cinco especies diferentes de mejillón pertenecientes al género Mytilus (M. Galloprovincialis, M. Edulis, M. Trossulus, M. Californianus y M. chilensis) y una especie perteneciente al género Perna (P. Canaliculus), sobre un total de 12 poblaciones distribuidas y recolectadas en diferentes costas. Se ha llevado a cabo el estudio de la variabilidad genética a través del análisis de cinco loci nucleares de copia única y tres secuencias de ADNs satélites altamente repetidos, estableciéndose relaciones filogenéticas entre dichas especies de mejillón. Asimismo,se efectuó un análisis de los posibles sucesos mutacionales que han tenido lugar a lo largo de la evolución de estos organismos, mediante el estudio de las secuencias nucleotídicas de los diferentes ADNs satélites. El análisis de las secuencias consenso obtenidas a partie de las de los ADNs satélites se emplearon para la construcción de filogenias,. Se ha evaluado y valorado el empleo de estos loci de copia única y de los ADNs satélites como posibles maracadores geneticos para la aplicación en productos manufacturados (conservas). Los resultados presentados contribuyen a clarificar el origen del género Mytilus.

[Abstract]: The DNA content of 10 species of bivalve molluscs from British Columbia coast was determined by image analysis, and the karyotypes of the horse clam “Tressus capax”, the bent-nose macoma “Macoma nasuta”, and the nuttall's mahogany clam “Nuttallia nuttallii” are described here for the first time. We also have analyzed the location of rDNA loci using a 28S-5.8S-18S probe in four of these species: “Mytilus californianus”, “M. trossulus”, “Macoma nasuta” and “N. nuttallii”. Results obtained report new data about cytogenetic characteristics of bivalve molluscs.