Jaime Castro

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.

Genetic structure of brown trout, salmo trutta l., at the southern limit of the distribution range of the anadromous form

Genetic variation at 33 protein loci was investigated in 41 wild brown trout populations from four river basins in Galicia (northwest Spain) to analyse the amount and distribution of genetic diversity in a marginal area, located in the distribution limit of the anadromous form of this species. The genetic diversity detected within populations (H between 0 and 6%) lies within the range quoted for this species in previous reports. The Miño, the most southern river basin analysed, showed a significantly lower genetic diversity and the highest genetic differentiation among the river basins studied. The hierarchical gene diversity analysis showed high population differentiation in a restricted area (GST = 27%), mostly due to differences among populations within basins (GSC = 22%). The reduction of GST observed when the isolated samples were excluded from the analysis (GST = 17%) showed the importance of habitat fragmentation on the heterogeneity detected. Gene flow among populations was comparatively evaluated by three indirect methods, which in general revealed low figures of absolute number of migrants per generation, slightly higher than 1. The gene flow among basins reflected a positive relationship with geographical distance. This trend was confirmed by the significant correlation observed between geographical and genetic distances, including all population pairs, which suggests a component of isolation by distance in brown trout genetic structure. Nevertheless, the nonsignificant intrabasin correlation demonstrates the complexity of genetic relationships among populations in this species. The model of genetic structure in brown trout is discussed in the light of the results obtained.

Differential stocking incidence in brown trout (Salmo trutta) populations from Northwestern Spain

Abstract An allozyme electrophoretic analysis was carried out to evaluate the impact of stocking, and to assess the amount and distribution of genetic variation among brown trout populations in Northwestern Spain. For this purpose, populations from both non-flowing (lagoon and reservoir) and flowing (rivers) waters, stocked and unstocked, were studied in comparison with the hatchery populations used for stocking in this region. Genetic variation was found at 11 of 35 loci analyzed. Detection of suitable genetic markers, especially the LDH-5 ∗ diagnostic locus, permitted us to monitor the incidence of stocking practices. The low viability of stocked individuals within river populations was remarkable, in spite of the long period and great intensity of repopulation. Only four individuals out of 197 analyzed were of hatchery origin. The absence of introgression detected in river populations was accounted for by the extreme Wahlund effect observed at the diagnostic locus LDH-5 ∗ . On the contrary, in non-flowing waters a large introgression was revealed. The longer the period of stocking, the larger the introgression detected. Gene diversity analysis demonstrated an important genetic differentiation among natural populations (G ST =0.273), mostly within drainages (86%), which shows an important microgeographical differentiation component. In sharp contrast the stocking hatcheries exhibited genetic homogeneity (G ST =0.014). Genetic distance (D=0.046) between indigenous and stocking groups apparently shows the presence of two divergent evolutionary lineages of brown trout. The levels of genetic variation were far greater in populations from non-flowing waters and stocking hatcheries as compared with river populations. This fact can be accounted for by the mixed origin of the former populations.

Characterization of an atypical NOR site polymorphism in brown trout (Salmo trutta) with Ag- and CMA3-staining, and fluorescent in situ hybridization

We have analyzed by Ag- and CMA3-staining, and rDNA fluorescent in situ hybridization an unusual NOR site polymorphism in a population of Salmo trutta from Mino basin (Northwestern Spain). A multichromosomal Ag-NOR distribution largely different from standard individuals both in the number and position of Ag-NORs, as well as in the pattern of activity, was revealed. Atypical Ag-NORs consist of rDNA genes, as evidenced by rDNA FISH, and are capable of constituting their own nucleolus. In spite of the large number of available NORs per individual, the mean number of Ag-NOR per cell was less than 3 in most individuals, which suggests a regulation mechanism to adjust the ribosomal production. The new rDNA clusters detected seem to be stably integrated, however, some characteristics of the NOR pattern observed suggest that this polymorphism is an unstable phenomenon.

Population analysis of an unusual NOR-site polymorphism in brown trout (Salmo trutta L.)

A population analysis of an unusual NOR-site polymorphism previously detected in brown trout (Salmo trutta L.) from North-western Spain was carried out in 225 individuals from 20 native populations from this area. The analysis performed has permitted us to reveal: (i) the ubiquity of this phenomenon in most river basins from NW Spain, 13 extra-NORs not observed in standard trout being detected; (ii) the rDNA constitution of all extra-NORs, as confirmed by CMA3-staining and rDNA-FISH, and their capability to constitute their own nucleolus; (iii) the constant location of extra-NORs within individuals and their stable transmission across generations; and (iv) the telomeric location of Ag-NORs, which were randomly distributed in the karyotype of S. trutta, mostly in the heterozygous condition. Repetitive sequences in the IGS or scattered along rDNA units, rather than chromosome rearrangements, could play an important role in the dispersion of NORs. The polymorphism described seems to have a single geographical origin, since a positive correlation was demonstrated between the degree of polymorphism and geographical distance to a central point of the river basin where the phenomenon showed a higher intensity. The distribution of NOR-site variation as compared with allozyme variation in the populations studied, showed significant statistical differences. Selection against high copy number, or mutation due to changes in NOR location, could be acting on this polymorphism to explain the observed distribution. The high dispersion of NOR-site variants within and among populations suggests the transposition phenomenon responsible for NOR jumping could be still active.

Localization of ribosomal genes in Pleuronectiformes using Ag‐, CMA3‐banding and in situ hybridization

In this paper we present the analysis of nucleolar organizer regions (NORs) in five species of the order Pleuronectiformes (Scophthalmus maximus, Scophthalmus rhombus, Platichthys flesus, Solea solea and Solea lascaris), a group of fish poorly studied cytogenetically. In spite of the small chromosome sizes, which characterize the karyotypes of this group, we implement both classical (Ag- and CMA3-staining) and molecular (in situ hybridization with a major rDNA probe) techniques for NOR location. NORs were localized in a single chromosome pair in all species studied and showed size variation within each species. Evidence of a NOR-site polymorphism was detected in Platichthys flesus and Scophthalmus maximus. NOR location was apparently the same within families (S. rhombus vs. S maximus, and S. solea vs. S. lascaris) but clearly differed among families, and supports the phylogenetic relationships proposed for the order Pleuronectiformes.

Allozymic evidence of parapatric differentiation of brown trout (Salmo trutta L.) within an Atlantic river basin of the Iberian Peninsula

The genetic variation of brown trout from Duero, one of the main Atlantic Iberian river basins, was assessed at 34 enzymatic loci in 62 native populations. A strong intrabasin differentiation was detected (GST = 0.46; range D: 0–0.066), mainly attributable to the existence of two divergent groups of populations within Duero: southern and northern groups. This divergence was mainly a consequence of the unequal distribution of *75 and *100 alleles at sMDH‐B1,2* isoloci, which were correlated with substantial differences in genetic diversity among regions. The Lower Course region (nearly fixed for the *100 allele) and Pisuerga River (nearly fixed for the *75 allele) showed lower heterozygosities (H ≈ 0.8%) in contrast with adjacent areas, which evidenced intermediate frequencies for both alleles and higher heterozygosities (H: 2.2–3.1%). Vicariance appeared as the more probable explanation for the significant positive correlation detected between genetic and geographical distances in Duero Basin. Genetic relationships with adjacent Iberian drainages indicate a close similarity between the southern group and Cantabric trout, whereas the northern group constitutes an ancient form from this basin. This study confirmed complex genetic relationships in brown trout from northwest Iberia, reasserting the existence of clines at several loci and for genetic diversity. The interaction between Cantabric and Duero trout, as well as the location of the limit of the anadromous form around the 42° N parallel, are both required to understand the genetic characteristics of brown trout from this area.

Gynogenesis assessment using microsatellite genetic markers in turbot ( Scophthalmus maximus)

Gynogenesis was assessed by different methods in 2 families of gynogenetic offspring in turbot (Scophthalmus maximus). Karyotype analysis in embryos and larvae demonstrated high accuracy in estimation of ploidy level, but performance was uneven given the low quality and number of plates obtained. The use of silver staining to estimate the number of nucleoli per nucleus resulted in a straightforward and easy method to evaluate the ploidy of the samples studied. However, the existence of a nucleolus organizer region polymorphism in turbot determined a small error in ploidy estimation, important when checking ploidy in specific individuals. The use of a set of 11 highly variable microsatellite loci proved to be a powerful method to confirm the exclusive maternal inheritance to gynogenetic offspring in turbot, with probabilities of detection of putative paternal genetic contribution above 99.99%.

Threatened freshwater pearl mussel Margaritifera margaritifera L. in NW Spain: low and very structured genetic variation in southern peripheral populations assessed using microsatellite markers

A genetic analysis of freshwater pearl mussel Margaritifera margaritifera populations from NW Spain, a peripheral area of its European distribution, was carried out using microsatellite markers. These populations were formerly reported as genetically differentiated on the basis of growth and longevity studies. Ten loci previously characterized in populations from central Europe were used to comparatively analyze the genetic variability at the southern edge of the species’ range. Iberian pearl mussel populations showed very low genetic variability and significant high genetic differentiation. Half of the total genetic diversity observed appeared to be distributed between populations, which suggested a highly structured adaptive potential in pearl mussel at the southern peripheral distribution of the species. Population distinctiveness was evidenced by assignment tests, which revealed a high accuracy of individual assignments to their population of origin. All data suggested low effective population size and major effects of genetic drift on population genetic structure. In order to avoid further loss of genetic variation in biologically distinctive populations from NW Spain, prioritization of genetic resources of this species is required for conservation and management.

Mitochondrial haplotype variability of brown trout populations from Northwestern Iberian Peninsula, a secondary contact area between lineages

Previous studies using mitochondrial markers and microsatellites revealed a new highly differentiated Atlantic lineage of brown trout in the Iberian Peninsula mainly confined to the Upper Duero Basin. Data suggest that Duero lineage (DU) hybridizes with previously described Atlantic (AT) lineages in that basin. However, nuclear and cytogenetic data suggest another hybrid zone between divergent lineages might be present in the Miño Basin, another Atlantic drainage located to the north. Here, we extend those studies by analyzing the mitochondrial haplotype variability of brown trout populations from northwestern Spain, a poorly studied area, to obtain an accurate picture of the distribution of DU. Results show that the DU haplotype is fixed in several isolated populations from the upper course of the Miño River and its main tributary the Sil River. DU haplotype co-occurs with two related AT haplotypes in the Miño Basin revealing a new secondary contact zone. The identification of both longtime stable populations of DU and several other populations showing introgression with widespread AT lineages will be useful for the management and conservation of such an endemic lineage.