Development and testing of a combined species SNP array for the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata).

SNP arrays are powerful tools for high-resolution studies of the genetic basis of complex traits, facilitating both selective breeding and population genomic research. The European seabass (Dicentrarchus labrax) and the gilthead seabream (Sparus aurata) are the two most important fish species for Mediterranean aquaculture. While selective breeding programmes increasingly underpin stock supply for this industry, genomic selection is not yet widespread. Genomic selection has major potential to expedite genetic gain, particularly for traits practically impossible to measure on selection candidates, such as disease resistance and fillet characteristics. The aim of our study was to design a combined-species 60 K SNP array for European seabass and gilthead seabream, and to test its performance on farmed and wild populations from numerous locations throughout the species range. To achieve this, high coverage Illumina whole-genome sequencing of pooled samples was performed for 24 populations of European seabass and 27 populations of gilthead seabream. This resulted in a database of ~20 million SNPs per species, which were then filtered to identify high-quality variants and create the final set for the development of the 'MedFish' SNP array. The array was then tested by genotyping a subset of the discovery populations, highlighting a high conversion rate to functioning polymorphic assays on the array (92% in seabass; 89% in seabream) and repeatability (99.4-99.7%). The platform interrogates ~30 K markers in each species, includes features such as SNPs previously shown to be associated with performance traits, and is enriched for SNPs predicted to have high functional effects on proteins. The array was demonstrated to be effective at detecting population structure across a wide range of fish populations from diverse geographical origins, and to examine the extent of haplotype sharing among Mediterranean farmed fish populations. In conclusion, the new MedFish array enables efficient and accurate high-throughput genotyping for genome-wide distributed SNPs for each fish species, and will facilitate stock management, population genomics approaches, and acceleration of selective breeding through genomic selection.

Dynamics of gene expression patterns during early development of the European seabass (Dicentrarchus labrax).

Larval and embryonic stages are the most critical period in the life cycle of marine fish. Key developmental events occur early in development and are influenced by external parameters like stress, temperature, salinity, and photoperiodism. Any failure may cause malformations, developmental delays, poor growth, and massive mortalities. Advanced understanding of molecular processes underlying marine larval development may lead to superior larval rearing conditions. Today, the new sequencing and bioinformatic methods allow transcriptome screens comprising messenger (mRNA) and microRNA (miRNA) with the scope of detecting differential expression for any species of interest. In the present study, we applied Illumina technology to investigate the transcriptome of early developmental stages of the European seabass (Dicentrarchus labrax). The European seabass, in its natural environment, is a euryhaline species and has shown high adaptation processes in early life phases. During its embryonic and larval phases the European seabass lives in a marine environment and as a juvenile it migrates to coastal zones, estuaries, and lagoons. Investigating the dynamics of gene expression in its early development may shed light on factors promoting phenotypic plasticity and may also contribute to the improvement and advancement of rearing methods of the European seabass, a species of high economic importance in European and Mediterranean aquaculture. We present the identification, characterization, and expression of mRNA and miRNA, comprising paralogous genes and differentially spliced transcripts from early developmental stages of the European seabass. We further investigated the detection of possible interactions of miRNA with mRNA.

Comparative analysis reveals that polyploidy does not decelerate diversification in fish.

While the proliferation of the species-rich teleost fish has been ascribed to an ancient genome duplication event at the base of this group, the broader impact of polyploidy on fish evolution and diversification remains poorly understood. Here, we investigate the association between polyploidy and diversification in several fish lineages: the sturgeons (Acipenseridae: Acipenseriformes), the botiid loaches (Botiidae: Cypriniformes), Cyprininae fishes (Cyprinidae: Cypriniformes) and the salmonids (Salmonidae: Salmoniformes). Using likelihood-based evolutionary methodologies, we co-estimate speciation and extinction rates associated with polyploid vs. diploid fish lineages. Family-level analysis of Acipenseridae and Botiidae revealed no significant difference in diversification rates between polyploid and diploid relatives, while analysis of the subfamily Cyprininae revealed higher polyploid diversification. Additionally, order-level analysis of the polyploid Salmoniformes and its diploid sister clade, the Esociformes, did not support a significantly different net diversification rate between the two groups. Taken together, our results suggest that polyploidy is generally not associated with decreased diversification in fish - a pattern that stands in contrast to that previously observed in plants. While there are notable differences in the time frame examined in the two studies, our results suggest that polyploidy is associated with different diversification patterns in these two major branches of the eukaryote tree of life.

Quantitative trait loci affecting morphology traits in gilthead seabream (Sparus aurata L.).

We report a quantitative trait loci (QTL) mapping study on 18 morphometric characters in gilthead seabream based on a total of 74 informative microsatellite markers genotyped in 409 offspring coming from 10 paternal half-sib families. Statistical analysis was carried out using a linear regression approach, and various suggestive and significant morphology QTL were detected in three (9, 21 and 25) of nine linkage groups examined. Fitting body weight as a covariate reduced the significance of some QTL but revealed three new QTL in other linkage groups (LG6 and LG10). Current results combined with those obtained from previous studies underline highly significant loci affecting overall growth and morphology in S. aurata.

Exploring neutral and adaptive processes in expanding populations of gilthead sea bream, Sparus aurata L., in the North-East Atlantic.

Recent studies in empirical population genetics have highlighted the importance of taking into account both neutral and adaptive genetic variation in characterizing microevolutionary dynamics. Here, we explore the genetic population structure and the footprints of selection in four populations of the warm-temperate coastal fish, the gilthead sea bream (Sparus aurata), whose recent northward expansion has been linked to climate change. Samples were collected at four Atlantic locations, including Spain, Portugal, France and the South of Ireland, and genetically assayed using a suite of species-specific markers, including 15 putatively neutral microsatellites and 23 expressed sequence tag-linked markers, as well as a portion of the mitochondrial DNA (mtDNA) control region. Two of the putatively neutral markers, Bld-10 and Ad-10, bore signatures of strong directional selection, particularly in the newly established Irish population, although the potential 'surfing effect' of rare alleles at the edge of the expansion front was also considered. Analyses after the removal of these loci suggest low but significant population structure likely affected by some degree of gene flow counteracting random genetic drift. No signal of historic divergence was detected at mtDNA. BLAST searches conducted with all 38 markers used failed to identify specific genomic regions associated to adaptive functions. However, the availability of genomic resources for this commercially valuable species is rapidly increasing, bringing us closer to the understanding of the interplay between selective and neutral evolutionary forces, shaping population divergence of an expanding species in a heterogeneous milieu.

Imprints from genetic drift and mutation imply relative divergence times across marine transition zones in a pan-European small pelagic fish (Sprattus sprattus).

Geographic distributions of most temperate marine fishes are affected by postglacial recolonisation events, which have left complex genetic imprints on populations of marine species. This study investigated population structure and demographic history of European sprat (Sprattus sprattus L.) by combining inference from both mtDNA and microsatellite genetic markers throughout the species' distribution. We compared effects from genetic drift and mutation for both genetic markers in shaping genetic differentiation across four transition zones. Microsatellite markers revealed significant isolation by distance and a complex population structure across the species' distribution (overall θ(ST)=0.038, P<0.01). Across transition zones markers indicated larger effects of genetic drift over mutations in the northern distribution of sprat contrasting a stronger relative impact of mutation in the species' southern distribution in the Mediterranean region. These results were interpreted to reflect more recent divergence times between northern populations in accordance with previous findings. This study demonstrates the usefulness of comparing inference from different markers and estimators of divergence for phylogeographic and population genetic studies in species with weak genetic structure, as is the case in many marine species.

Quantitative trait loci for body growth and sex determination in the hermaphrodite teleost fish Sparus aurata L.

Gilthead sea bream (Sparus aurata L.) is an important marine fish in Mediterranean aquaculture. Sex determination by age and/or body weight is a critical life-history trait, the genetic basis for which is largely unknown in this sequential hermaphrodite species. Herein, we performed a partial genome scan to map quantitative trait loci (QTL) affecting body weight and sex using 74 informative microsatellite markers from 10 paternal half-sib families to construct nine linkage groups (LG). In total, four growth-related QTL (two chromosome-wide and two genome-wide) and six QTL related to sex determination (three pairs in three different LGs) were detected (two chromosome-wide and one genome-wide). The proportion of phenotypic variation explained by the body-weight QTL ranged from 9.3% to 17.2%, showing their potential for use in marker-assisted selection. The results obtained offer solid ground to investigate the structure and function of the genomic regions involved in the mechanisms of sex reversal.

Linkage mapping, comparative genome analysis, and QTL detection for growth in a non-model teleost, the meagre Argyrosomus regius, using ddRAD sequencing.

Meagre (Argyrosomus regius), is a benthopelagic species rapidly emerging in aquaculture, due to its low food to biomass conversion rate, good fillet yield and ease of production. Tracing a species genomic background along with describing the genetic basis of important traits can greatly influence both conservation strategies and production perspectives. In this study, we employed ddRAD sequencing of 266 fish from six F1 meagre families, to construct a high-density genetic map comprising 4529 polymorphic SNP markers. The QTL mapping analysis provided a genomic appreciation for the weight trait identifying a statistically significant QTL on linkage group 15 (LG15). The comparative genomics analysis with six teleost species revealed an evolutionarily conserved karyotype structure. The synteny observed, verified the already well-known fusion events of the three-spine stickleback genome, reinforced the evidence of reduced evolutionary distance of Sciaenids with the Sparidae family, reflected the evolutionary proximity with Dicentrarchus labrax, traced several putative chromosomal rearrangements and a prominent putative fusion event in meagre's LG17. This study presents novel elements concerning the genome evolutionary history of a non-model teleost species recently adopted in aquaculture, starts to unravel the genetic basis of the species growth-related traits, and provides a high-density genetic map as a tool that can help to further establish meagre as a valuable resource for research and production.

Genetic diversity of the imperilled bath sponge Spongia officinalis Linnaeus, 1759 across the Mediterranean Sea: patterns of population differentiation and implications for taxonomy and conservation.

The Mediterranean bath sponge Spongia officinalis is an iconic species with high socio-economic value and precarious future owing to unregulated harvesting, mortality incidents and lack of established knowledge regarding its ecology. This study aims to assess genetic diversity and population structure of the species at different geographical scales throughout its distribution. For this purpose, 11 locations in the Eastern Mediterranean (Aegean Sea), Western Mediterranean (Provence coast) and the Strait of Gibraltar were sampled; specimens were analysed using partial mitochondrial cytochrome oxidase subunit I (COI) sequences, along with a set of eight microsatellite loci. According to our results (i) no genetic differentiation exists among the acknowledged Mediterranean morphotypes, and hence, S. officinalis can be viewed as a single, morphologically variable species; (ii) a notable divergence was recorded in the Gibraltar region, indicating the possible existence of a cryptic species; (iii) restriction to gene flow was evidenced between the Aegean Sea and Provence giving two well-defined regional clusters, thus suggesting the existence of a phylogeographic break between the two systems; (iv) low levels of genetic structure, not correlated to geographical distance, were observed inside geographical sectors, implying mechanisms (natural or anthropogenic) that enhance dispersal and gene flow have promoted population connectivity; (v) the genetic diversity of S. officinalis is maintained high in most studied locations despite pressure from harvesting and the influence of devastating epidemics. These findings provide a basis towards the effective conservation and management of the species.

Mitochondrial DNA reveals the genealogical history of the snake-eyed lizards (Ophisops elegans and O. occidentalis) (Sauria: Lacertidae).

The snake-eyed lizards of the genus Ophisops (Lacertidae) have been through a series of taxonomical revisions, but still their phylogenetic relationships remain uncertain. In the present study we estimate the phylogeographic structure of O. elegans across its distributional range and we evaluate the relationships between O. elegans and the sympatric, in North Africa, species O. occidentalis, using partial mtDNA sequences (16S rRNA, COI, and cyt b). All phylogenetic analyses produced topologically identical trees where extant populations of O. elegans and O. occidentalis were found polyphyletic. Taking into account all the potential causes of polyphyly (introgressive hybridization, incomplete lineage sorting, and imperfect taxonomy) we suggest the inaccurate taxonomy as the most likely explanation for the observed pattern. Our results stress the need for re-evaluation of the current taxonomical status of these species and their subspecies. Furthermore, our biogeographic analyses and the estimated time of divergences suggest a late Miocene diversification within these species, where the present distribution of O. elegans and O. occidentalis was the result of several dispersal and vicariant events, which are associated with climatic oscillations (the late Miocene aridification of Asia and northern Africa) and paleogeographic barriers of late Miocene and Pliocene period.

A combined AFLP and microsatellite linkage map and pilot comparative genomic analysis of European sea bass Dicentrarchus labrax L.

European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) sustains a regional fishery and is commonly farmed in the Mediterranean basin, but has not undergone much long-term genetic improvement. An updated genetic linkage map of the European sea bass was constructed using 190 microsatellites, 176 amplified fragment length polymorphisms and two single nucleotide polymorphisms. From the 45 new microsatellite markers (including 31 type I markers) reported in this study, 28 were mapped. A total of 368 markers were assembled into 35 linkage groups. Among these markers, 28 represented type I (coding) markers, including those located within the peptide Y, SOX10, PXN1, ERA and TCRB genes (linkage groups 1, 7, 16, 17 and 27 respectively). The sex-averaged map spanned 1373.1 centimorgans (cM) of the genome. The female map measured 1380.0 cM, whereas the male map measured 1046.9 cM, leading to a female-to-male (F:M) recombination rate ratio of 1.32:1. The intermarker spacing of the second-generation linkage map of the European sea bass was 3.67 cM, which is smaller than that of the first-generation linkage map (5.03 cM). Comparative mapping of microsatellite flanking regions was performed with five model teleosts and this revealed a high percentage (33.6%) of evolutionarily conserved regions with the three-spined stickleback.

Muscle and liver transcriptome characterization and genetic marker discovery in the farmed meagre, Argyrosomus regius.

Meagre (Argyrosomus regius), a teleost fish of the family Sciaenidae, is part of a group of marine fish species considered new for Mediterranean aquaculture representing the larger fish cultured in the region. Meagre aquaculture started ~25years ago in West Mediterranean, and the supply of juveniles has been dominated by few hatcheries. This fact has raised concerns on possible inbreeding, urging the need for genetic information on the species and for an assessment of the polymorphisms found in the genome. To that end we characterized the muscle and liver transcriptome of a pool of meagre individuals, from different families and phenotypic size, to obtain a backbone that can support future studies regarding physiology, immunology and genetics of the species. The assembled transcripts were assigned to a wide range of biological processes including growth, reproduction, metabolism, development, stress and behavior. Then, to infer its genetic diversity and provide a catalogue of markers for future use, we scanned the reconstructed transcripts for polymorphic genetic markers. Our search revealed a total of 42,933 high quality SNP and 20,581 STR markers. We found a relatively low rate of polymorphism in the transcriptome that may indicate that inbreeding has taken place. This study has led to a catalogue of genetic markers at the expressed part of the genome and has set the ground for understanding growth and other traits of interest in meagre.

The transcriptomic signature of different sexes in two protogynous hermaphrodites: Insights into the molecular network underlying sex phenotype in fish.

Sex differentiation is a puzzling problem in fish due to the variety of reproductive systems and the flexibility of their sex determination mechanisms. The Sparidae, a teleost family, reflects this remarkable diversity of sexual mechanisms found in fish. Our aim was to capture the transcriptomic signature of different sexes in two protogynous hermaphrodite sparids, the common pandora Pagellus erythrinus and the red porgy Pagrus pagrus in order to shed light on the molecular network contributing to either the female or the male phenotype in these organisms. Through RNA sequencing, we investigated sex-specific differences in gene expression in both species' brains and gonads. The analysis revealed common male and female specific genes/pathways between these protogynous fish. Whereas limited sex differences found in the brain indicate a sexually plastic tissue, in contrast, the great amount of sex-biased genes observed in gonads reflects the functional divergence of the transformed tissue to either its male or female character. Α common "crew" of well-known molecular players is acting to preserve either sex identity of the gonad in these fish. Lastly, this study lays the ground for a deeper understanding of the complex process of sex differentiation in two species with an evolutionary significant reproductive system.

Characterization of nine polymorphic microsatellite markers in sprat (Sprattus sprattus L.).

Nine polymorphic microsatellites were isolated from sprat (Sprattus sprattus) using a microsatellite enrichment protocol and selective hybridization with a biotinylated (AC)(12) probe. The loci showed different variation patterns in a Baltic Sea population (44 individuals) with mean number of alleles at 12.7 and mean observed heterozygosity at 0.78. These microsatellite loci are expected to be used for taxonomic considerations in sprat, stock differentiation and population genetic analysis.

Mitochondrial phylogeography of Rana (Pelophylax) populations in the Eastern Mediterranean region.

Phylogenetic relationships in the western fraction of Rana (Pelophylax) have not been resolved till now, even though several works have been devoted to the subject. Here, we infer phylogenetic relationships among the species distributed in the area of the Eastern Mediterranean, comparing partial mitochondrial DNA sequences for the cytochrome b and 16S rRNA genes. The obtained molecular data clearly indicate that Western Palearctic water frogs underwent a basal radiation into at least 3 major lineages (the perezi, the lessonae, and the rindibunda/bedriagae lineages) advocating an upper Miocene speciation. Moreover, we consider that within the rindibunda/bedriagae lineage, R. (P.) ridibunda, R. (P.) epeirotica, R. (P.) cretensis, R. (P.) bedriagae, R. (P.) cerigensis and R. (P.) kurtmuelleri were differentiated from a common ancestor through a series of vicariant and dispersal events, during the last approximately 5Mya, even though the specific rank of some taxa may be questionable, such as R. cerigensis in respect with R. bedriagae and R. kurtmuelleri in respect with R. ridibunda.

Biotype status and genetic polymorphism of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in Greece: mitochondrial DNA and microsatellites.

The genetic polymorphism and the biotype identity of the tobacco whitefly Bemisia tabaci (Gennadius) have been studied in population samples taken from different localities within Greece from cultivated plants growing in greenhouses or in open environments and from non-cultivated plants. Two different approaches were used: sequencing of the mitochondrial cytochrome oxidase I (mtCOI) gene and genotyping using microsatellite markers. Analyses of the mtCOI sequences revealed a high homogeneity between the Greek samples which clustered together with Q biotype samples that had been collected from other countries. When genetic polymorphism was examined using six microsatellite markers, the Greek samples, which were all characterized as Q biotype were significantly differentiated from each other and clustered into at least two distinct genetic populations. Moreover, based on the fixed differences revealed by the mtCOI comparison of known B. tabaci biotype sequences, two diagnostic tests for discriminating between Q and B and non-Q/non-B biotypes were developed. Implementation of these diagnostic tools allowed an absence of the B biotype and presence of the Q biotype in the Greek samples to be determined.

Phylogenetic relationships and evolutionary history of snake-eyed skink Ablepharus kitaibelii (Sauria: Scincidae).

Sequence data derived from two mitochondrial markers, 16S rRNA and cytochrome b genes, were used to infer the phylogenetic relationships of 38 populations of the snake-eyed skinks of the genus Ablepharus with emphasis on A. kitaibelii from Greece and Turkey. The partition-homogeneity tests indicated that the combined data set was homogeneous, and maximum-parsimony, maximum-likelihood, and Bayesian analyses produced topologically identical trees that revealed a well-resolved phylogeny. All species except A. kitaibelii form monophyletic units. The latter species appears paraphyletic with respect to A. budaki and A. chernovi with populations clustering into two distinct clades. A. chernovi and A. budaki, which have recently been raised to species status, were confirmed as genetically distinct forms. We used sequence divergence and paleogeographic history of the Aegean region to reconstruct a biogeographic evolutionary scenario for A. kitaibelii.

Molecular phylogeny of north mediterranean freshwater barbs (genus Barbus: cyprinidae) inferred from cytochrome b sequences: biogeographic and systematic implications.

We investigated phylogenetic relationships among north Mediterranean species of the genus Barbus using sequences of the cytochrome b gene. Our results indicate that the species belong to two major clades that are consistent with those previously defined from morphological features. The first clade includes species ranging from France to the Black Sea. In this clade, there is a well-supported monophyletic group of large-sized fluvio-lacustrine barbs; however, the monophyly of the small-sized rheophilic species is not clear. The second clade comprises species found in Spain, Greece, and Asia Minor and probably represents the oldest group present in the north Mediterranean rivers. In general, there is good concordance between geography and phylogenetic relationships. These results are compared to those from previous morphological- and allozyme-based studies and demonstrate widespread discordance and polyphyly in the traditional taxonomy of the genus Barbus. This study is one of the first reporting the phylogenetic and biogeographic relationships of a genus that is widely distributed in European rivers and contains species that are a major component of the European ichthyofauna.

Multiple origins of polyploidy in the phylogeny of southern African barbs (Cyprinidae) as inferred from mtDNA markers.

The cyprinid genus Barbus, with more than 800 nominal species, is an apparently polyphyletic assemblage to which a number of unrelated species, groups and/or assemblages have been assigned. It includes species that exhibit three different ploidy levels: diploid, tetraploid and hexaploid. Several lineages of the family Cyprinidae constitute a major component of the African freshwater ichthyofauna, having about 500 species, and fishes assigned to the genus 'Barbus' have the most species on the continent. We used complete sequences of the mitochondrial cytochrome b gene in order to infer phylogenetic relationships between diploid, tetraploid and hexaploid species of 'Barbus' occurring in southern Africa, the only region where representatives of all of the three ploidy levels occur. The results indicate that most of the lineages are incorrectly classified in the genus 'Barbus'. The southern African tetraploids probably originated from southern African diploids. They constitute a monophyletic group distinct from tetraploids occurring in the Euro-Mediterranean region (Barbus sensu stricto). The 'small' African diploid species seem to be paraphyletic, while the 'large' African hexaploid barbs species are of a single, recent origin and form a monophyletic group. The evidence of multiple, independent origins of polyploidy occurring in the African cyprinine cyprinids thus provides a significant contribution to the knowledge on the systematic diversity of these fishes, and warrants a thorough taxonomic reorganization of the genus.

Phylogeny and biogeography of the family Cyprinidae in the Middle East inferred from cytochrome b DNA- evolutionary significance of this region.

The phylogenetic relationships of cyprinid species from the Middle East and neighboring biogeographical areas were investigated using cytochrome b sequence variation in order to test hypotheses that consider the Middle Eastern area as an important interchange area or a center of speciation for the freshwater fauna. A total of 62 cyprinid species were analyzed over the complete cytochrome b fragment (1140 bp); 28 belong to the Leuciscinae subfamily and 34 to the Cyprininae. All the Leuciscinae lineage fish recorded in the Middle East are also found in Europe, which was interpreted as an important Palearctic influence in the Middle Eastern ichthyofauna consistent with the Lago Mare dispersion. However, it has also been suggested that several Danube species have their origins in the Middle East. In contrast, the Cyprininae subfamily showed three highly divergent lineages, one shared with the Euro-Mediterranean area (Barbus/Luciobarbus genus) relict of the Lago Mare dispersion, one shared with Africa (Carasobarbus/Varicorhinus subgenus), and the third shared with Asia (Garra genus). Furthermore, clades observed in the phylogenetic reconstructions are not consistent with morphometric or karyological data and disagree with previous taxonomic assumptions. Lastly, the dispersion history in the Middle East of this subfamily appears much more complicated and ancient than that of the Leuciscinae. However, taking into account Cyprininae and Leuciscinae distribution, the Middle East appears more like an important interchange area for the freshwater ichtyofauna than a center of speciation.