Carmelo Ruiz-Rejón

The molecular diagnosis of Marteilia refringens and differentiation between Marteilia strains infecting oysters and mussels based on the rDNA IGS sequence.

Marteilia refringens is a paramyxean parasite which infects the flat oyster Ostrea edulis and mussels (Mytilus galloprovincialis), where it has been attributed to a separate species, Marteilia maurini, by several authors. Doubts persist though as to the existence or not of two species of Marteilia in Europe. We have devised a molecular method for the diagnosis of M. refringens based on 358 bp nested-PCR of the rDNA intergene spacer (rDNA IGS) which is capable of detecting 0.5 fg of M. refringens DNA. Molecular characterization of this spacer indicates that the Marteilia parasites which infect oysters and mussels are two different strains of the same species.

Identification of Marteilia refringens infecting the razor clam Solen marginatus by PCR and in situ hybridization.

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. It is believed to have a complex life-cycle involving several hosts. In this study, we applied molecular approaches to identify this parasite in samples of the razor clam Solen marginatus from the south west coast of Spain. We used a PCR assay to amplify a fragment of the IGS rDNA region. PCR products were sequenced and the phylogenetic affinity of the sequences was determined. In situ hybridization analysis showed tissue distribution and presence of different developmental stages of the parasite in the digestive diverticula epithelium, which suggested a true parasitism in these individuals. This is the first report of the occurrence of M. refringens in the razor clam S. marginatus in the south Atlantic. The methodology described herein may be useful for accurate identification of the parasite strain in different hosts and thus provide valuable information for marteiliosis control programmes.

Detection of Marteilia refringens using nested PCR and in situ hybridisation in Chamelea gallina from the Balearic Islands (Spain).

In the course of a histopathological survey performed to discover the cause of mass mortality of the striped clam Chamelea gallina in the Balearic Islands (Spain, Mediterranean Sea), we detected a Marteilia-like parasite in 3 clams. Molecular methods were applied to identify the parasite. DNA extracted from a paraffin block was used to carry out a PCR assay for Marteilia refringens detection based on a rDNA sequence of the parasite (the intergenic spacer of ribosomal genes, IGS). The nucleotide sequence of the IGS amplified fragment and the positive signal obtained by in situ hybridisation analysis with a M. refringens-specific probe allowed us to confirm the presence of this parasite in the digestive gland tissue of C. gallina.

Validation and comparison of microsatellite markers derived from Senegalese sole (Solea senegalensis, Kaup) genomic and expressed sequence tags libraries

In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic‐DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST‐SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST‐SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST‐SSRs, as expected. Within the EST‐SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular‐assisted breeding in this species.

Polyploidy, the major speciation mechanism in Muscari subgenus Botryanthus in the Iberian Peninsula

Currently, three species ofMuscari subg. Botryanthus are recognized in the Iberian Peninsula: two diploids (2n = 18), M. atlanticum and M. cazorlanum, and one morphologically variable species with three different ploidy levels, M. neglectum (2n = 36, 45, 54). We have made a comparative study of numerous Iberian populations to clarify the taxonomy and evolution of this group. To this end we carried out morphological and cytogenetic analyses, and phylogenetic analysis of the internal transcribed spacers of nuclear ribosomal DNA. Comparative and UPGMA analyses of the morphological characteristics show that the different ploidy levels of M. neglectum represent different species. We describe the pentaploid and hexaploid levels as two new species, M. olivetorum (2n = 45) and M. baeticum (2n = 54), each with an exclusive combination of morphological characters and a characteristic ecological behavior pattern. Phylogenetic study of ITS shows that the two new species are not autopolyploids from M. neglectum but allopolyploids. These findings are supported by the additivity of the three ITS variants found in M. olivetorum with the ITS of M. neglectum and M. baeticum, and also by morphology. Possible parents for both new species are proposed. Absence of homogenization between homeologous M olivetorum nrDNA loci is explained by the absence of sexual reproduction and by nucleolar dominance, indicating that this is a recent species.

First Haploid Genetic Map Based on Microsatellite Markers in Senegalese Sole (Solea senegalensis, Kaup 1858)

The Senegalese sole (Solea senegalensis, Kaup 1858) is a flatfish species of great value for aquaculture. In this study, we develop the first linkage map in this species based on microsatellite markers characterized from genomic DNA libraries and EST databases of Senegalese sole and from other flatfish species. Three reference gynogenetic families were obtained by chromosome-manipulation techniques: two haploid gynogenetics, used to assign and order microsatellites to linkage groups and another diploid gynogenetic family, used for estimating marker–centromere distances. The consensus map consists of 129 microsatellites distributed in 27 linkage groups (LG), with an average density of 4.7 markers per LG and comprising 1,004 centimorgans (cM). Additionally, 15 markers remained unlinked. Through half-tetrad analysis, we were able to estimate the centromere distance for 81 markers belonging to 24 LG, representing an average of 3 markers per LG. Comparative mapping was performed between flatfish species LG and model fish species chromosomes (stickleback, Tetraodon, medaka, fugu and zebrafish). The usefulness of microsatellite markers and the genetic map as tools for comparative mapping and evolution studies is discussed.

Analysis of Mitochondrial and Nuclear DNA Markers in Old Museum Sturgeons Yield Insights About the Species Existing in Western Europe: A. sturio, A. naccarii and A. oxyrinchus

Today, with all the sturgeon species almost disappearing all over the world, it is necessary to undertake their recovery under the programs for the conservation of genetic resources. The complete absence of these fish from most rivers increases the difficulties in carrying out such programs, hampering the genetic identification of specimens and their correct species assignment. However, with the development of new and reliable molecular genetic techniques, many studies such as this are yielding insights concerning the sturgeon species that inhabited European rivers in the past. In the last few years, our group has developed forensic techniques to isolate DNA from ancient sturgeon specimens preserved in museums. These DNA samples have been the subject of various analyses conducted on several nuclear and mitochondrial DNA markers. The combined use of both types of markers has provided accurate genetic identification of these specimens and has overcome the problem of misinterpretation caused by hybridization and introgression. Here, we show that, in addition to Acipenser sturio (the only species previously believed to inhabit the rivers of Western Europe), two other species, Acipenser naccarii and Acipenser oxyrinchus lived in these rivers. Thus, we have found evidence for the presence of A. naccarii in the Guadalquivir river in the Iberian Peninsula and in some rivers in Italy from the Tyrrhenian/Ligurian side, as well as for the presence of A. oxyrinchus in the Ebro river in Spain. Our studies clarify the distribution of sturgeon species in the Western Mediterranean and open new perspectives for recovery plans.

Contribution to the taxonomy and phylogeny of Sarcocapnos DC. (Fumariaceae)

Abstract. To solve problems concerning the status of the taxa described in the genus Sarcocapnos, we have conducted a study using morphological, pollen morphology (light microscopy), cytogenetic and molecular techniques. Focusing on the last technique, we have sequenced ITS-1 and ITS-2 of nuclear rDNA. The species differ basically according to 5 morphological traits (leaf shape, flower spur, corolla colour, corolla size, and crest of the stigmatic surface). The cytogenetic analyses indicated n=16 to be the standard chromosome number. The ITS analyses showed that the genus is monophyletic, defining two main well-supported clades, one containing S. saetabensis and S. enneaphylla, and one containing the rest of the species. In this second clade, S. speciosa, S. pulcherrima, and S. baetica subsp. ardalii are related, as are S. integrifolia, S. crassifolia subsp. crassifolia, and S. crassifolia subsp. atlantis; S. baetica subsp. baetica forms a trichotomy with the foregoing groups. S. speciosa is shown to be a species separate from S. crassifolia subsp. crassifolia, as in the case of S. baetica with respect to S. integrifolia. Palynologically, the parameters used enabled us to establish clear differences between the taxa, often corroborating the macromorphological and genetic data. The flower spur has been reduced several times in different groups of the genus, for which the classifications established on the basis of this trait are paraphyletic.

Long-term monitoring of B-chromosome invasion and neutralization in a population of Prospero autumnale (Asparagaceae): MONITORING B CHROMOSOME INVASION

B chromosomes have been reported in about 15% of eukaryotes, but long‐term dynamics of B chromosomes in a single natural population has rarely been analyzed. Prospero autumnale plants collected in 1981 and 1983 at Cuesta de La Palma population had shown the presence of B chromosomes. We analyze here seven additional samples collected between 1987 and 2015, and show that B frequency increased significantly during the 1980s and showed minor fluctuations between 2005 and 2015. A mother–offspring analysis of B chromosome transmission, at population level, showed significant drive on the male side (kB = 0.65) and significant drag on the female side (kB = 0.33), with average B transmission rate being very close to the Mendelian rate (0.5). No significant effects of B chromosomes were observed on a number of vigor and fertility‐related traits. Within a parasite/host framework, these results suggest that B chromosomes’ drive on the male side is the main pathway for B chromosome invasion, whereas B chromosome drag on the female side might be the main manifestation of host genome resistance in this species. Prospero autumnale thus illuminates a novel evolutionary pathway for B chromosome neutralization by means of a decrease in B transmission through the nondriving sex.