Accelerated Diversification Explains the Exceptional Species Richness of Tropical Characoid Fishes.

The Neotropics harbor the most species-rich freshwater fish fauna on the planet, but the timing of that exceptional diversification remains unclear. Did the Neotropics accumulate species steadily throughout their long history, or attain their remarkable diversity recently? Biologists have long debated the relative support for these museum and cradle hypotheses, but few phylogenies of megadiverse tropical clades have included sufficient taxa to distinguish between them. We used 1288 ultraconserved element loci spanning 293 species, 211 genera, and 21 families of characoid fishes to reconstruct a new, fossil-calibrated phylogeny and infer the most likely diversification scenario for a clade that includes a third of Neotropical fish diversity. This phylogeny implies paraphyly of the traditional delimitation of Characiformes because it resolves the largely Neotropical Characoidei as the sister lineage of Siluriformes (catfishes), rather than the African Citharinodei. Time-calibrated phylogenies indicate an ancient origin of major characoid lineages and reveal a much more recent emergence of most characoid species. Diversification rate analyses infer increased speciation and decreased extinction rates during the Oligocene at around 30 Ma during a period of mega-wetland formation in the proto-Orinoco-Amazonas. Three species-rich and ecomorphologically diverse lineages (Anostomidae, Serrasalmidae, and Characidae) that originated more than 60 Ma in the Paleocene experienced particularly notable bursts of Oligocene diversification and now account collectively for 68% of the approximately 2150 species of Characoidei. In addition to paleogeographic changes, we discuss potential accelerants of diversification in these three lineages. While the Neotropics accumulated a museum of ecomorphologically diverse characoid lineages long ago, this geologically dynamic region also cradled a much more recent birth of remarkable species-level diversity. [Biodiversity; Characiformes; macroevolution; Neotropics; phylogenomics; ultraconserved elements.].

Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish.

BACKGROUND: Eukaryote genomes frequently harbor supernumerary B chromosomes in addition to the "standard" A chromosome set. B chromosomes are thought to arise as byproducts of genome rearrangements and have mostly been considered intraspecific oddities. However, their evolutionary transcendence beyond species level has remained untested. RESULTS: Here we reveal that the large metacentric B chromosomes reported in several fish species of the genus Astyanax arose in a common ancestor at least 4 million years ago. We generated transcriptomes of A. scabripinnis and A. paranae 0B and 1B individuals and used these assemblies as a reference for mapping all gDNA and RNA libraries to quantify coverage differences between B-lacking and B-carrying genomes. We show that the B chromosomes of A. scabripinnis and A. paranae share 19 protein-coding genes, of which 14 and 11 were also present in the B chromosomes of A. bockmanni and A. fasciatus, respectively. Our search for B-specific single-nucleotide polymorphisms (SNPs) identified the presence of B-derived transcripts in B-carrying ovaries, 80% of which belonged to nobox, a gene involved in oogenesis regulation. Importantly, the B chromosome nobox paralog is expressed > 30× more than the A chromosome paralog. This indicates that the normal regulation of this gene is altered in B-carrying females, which could potentially facilitate B inheritance at higher rates than Mendelian law prediction. CONCLUSIONS: Taken together, our results demonstrate the long-term survival of B chromosomes despite their lack of regular pairing and segregation during meiosis and that they can endure episodes of population divergence leading to species formation.

Chromosomal Instability and Origin of B Chromosomes in the Amazonian Glass Tetra Moenkhausia oligolepis (Günther, 1864) (Characiformes, Characidae).

B chromosomes occur in different species of the small characid fishes of the genus Moenkhausia. These supernumerary elements, that do not recombine with chromosomes of the standard A complement and follow their own evolutionary mechanism vary in number, morphology, and distribution. Here, we show karyotypic data of individuals of 2 populations of Moenkhausia oligolepis of the Brazilian Amazon (Pedro Correia and Taboquinha streams, Tocantins river basin), both with a diploid number of 50 chromosomes and karyotypic formula of 10m + 32sm + 8a. In addition to the normal complement, we also observed the occurrence of B chromosomes in the 2 populations with intra- and interindividual variation ranging from 0 to 10 Bs, independent of sex. The C-banding pattern evidenced heterochromatic blocks located mainly in the pericentromeric region of the chromosomes, while the B chromosomes appeared euchromatic. Silver-stained nucleolus organizer regions were identified in multiples sites, and some of these blocks were positive when stained with chromomycin A3. The karyotype analysis and the application of whole-chromosome painting in populations of M. oligolepis reinforce the conservation of the basal diploid number for the genus, as well as the evolutionary tendency in these fishes to carry B chromosomes. Both populations turned out to be in different stages of stability and expansion of their B chromosomes. We further suggest that the origin of these chromosomes is due to the formation of isochromosomes. Here, we identified a pair of complement A chromosomes involved in this process.

Comparative eye and liver differentially expressed genes reveal monochromatic vision and cancer resistance in the shortfin mako shark (Isurus oxyrinchus).

The shortfin mako, Isurus oxyrinchus is an oceanic pelagic shark found worldwide in tropical and subtropical waters. However, the understanding of its biology at molecular level is still incipient. We sequenced the messenger RNA isolated from eye and liver tissues. De novo transcriptome yielded a total of 705,940 transcripts. A total of 3774 genes were differentially expressed (DEGs), with 1612 in the eye and 2162 in the liver. Most DEGs in the eye were related to structural and signaling functions, including nonocular and ocular opsin genes, whereas nine out of ten most overexpressed genes in the liver were related to tumor suppression, wound healing, and human diseases. Furthermore, DEGs findings provide insights on the monochromatic shark vision and a repertory of cancer-related genes, which may be insightful to elucidate shark resistance to cancer. Therefore, our results provide valuable sequence resources for future functional and population studies.

Molecular and morphological diversity in species of Kronichthys (Teleostei, Loricariidae) from Atlantic coastal rivers of Brazil.

The Neotropical catfish genus Kronichthys contains three species distributed along coastal rivers of southern and southeastern Brazil. Although phylogenetic hypotheses are available, the molecular and morphological diversity and species boundaries within the genus remain unexplored. In this study, the authors generated mitochondrial data for 90 specimens combined with morphometric and meristic data to investigate species diversity, species boundaries and putative morphological signatures in Kronichthys. Phylogenetic and species delimitation results clearly show the presence of four genetic lineages, three within Kronichthys heylandi along the coast from Rio de Janeiro to southern São Paulo and a single lineage encompassing both the nominal species Kronichthys lacerta and Kronichthys subteres from the Ribeira de Iguape basin to Santa Catarina in southern Brazil. Nonetheless, morphological data show overlapped ranges in morphometrics and a definition of only two morphotypes, with clear phenotypic differences in the teeth number: K. heylandi differs from K. subteres + K. lacerta by the higher number of premaxillary teeth (30-52 vs. 19-28) and higher number of dentary teeth (28-54 vs. 17-28). Headwater captures and connections of paleodrainages because of sea-level fluctuations represent the two major biogeographic processes promoting species diversification and lineage dispersal of Kronichthys in the Atlantic coastal range of Brazil.

Molecular cytogenetic analysis and the establishment of a cell culture in the fish species Hollandichthys multifasciatus (Eigenmann & Norris, 1900) (Characiformes, Characidae).

Hollandichthys is a fish genus of the family Characidae that was until recently considered to be monotypic, with cytogenetic, morphological, and molecular data being restricted to a few local populations. In the present study, the karyotype of a population of Hollandichthys multifasciatus was analyzed using classical and molecular cytogenetic approaches for the investigation of potential markers that could provide new perspectives on the cytotaxonomy. H. multifasciatus presented a diploid number of 2n=50 chromosomes and a karyotype formula of 8m+10sm+32st. A single pair of chromosomes presented Ag-NORs signals, which coincided with the 18S rDNA sites visualized by FISH, whilst the 5S rDNA sequences were mapped in two chromosome pairs. The distribution of the U snRNA genes was mapped on the Hollandichthys chromosomes for the first time, with the probes revealing the presence of the U1 snDNA on the chromosomes of pair 20, U2 on pairs 6 and 19, U4 on pair 16, and U6 on the chromosomes of pair 11. The results of the present study indicated karyotypic differences in comparison with the other populations of H. multifasciatus studied previously, reinforcing the need for further research to identify isolated populations or the potential existence of cryptic Hollandichthys species.

Establishment of rapid and non-invasive protocols to identify B-carrying individuals of Psalidodon paranae.

Supernumerary, or B, chromosomes are present in several eukaryotes, including characid fish of the genus Psalidodon. Notably, Psalidodon paranae carries the most studied B chromosome variant, a macro-B chromosome. The origin of this element was determined to be an isochromosome; however, data regarding its inheritance remain unavailable due to methodological barriers such as the lack of an efficient, non-invasive, and rapid protocol for identifying B-carrying individuals that would enable the design of efficient crossing experiments. Thus, in this study, we primarily aimed was to develop two non-invasive and fast (approximately 2 h) methods to identify the presence of B chromosomes in live specimens of P. paranae based on satellite DNA (satDNA) sequences known to be present in this element. The methods include fluorescence in situ hybridization in interphase nuclei and relative gene quantification of satDNAs using quantitative polymerase chain reaction. Our results reveal the efficiency of quick-fluorescence in situ hybridization and quantitative polymerase chain reaction for identifying B-carrying individuals using the proposed satDNA sequences and open up new possibilities to study B chromosomes.

Meiotic self-pairing of the Psalidodon (Characiformes, Characidae) iso-B chromosome: A successful perpetuation mechanism.

B chromosomes are non-essential additional genomic elements present in several animal and plant species. In fishes, species of the genus Psalidodon (Characiformes, Characidae) harbor great karyotype diversity, and multiple populations carry different types of non-essential B chromosomes. This study analyzed how the dispensable supernumerary B chromosome of Psalidodon paranae behaves during meiosis to overcome checkpoints and express its own meiosis-specific genes. We visualized the synaptonemal complexes of P. paranae individuals with zero, one, or two B chromosomes using immunodetection with anti-medaka SYCP3 antibody and fluorescence in situ hybridization with a (CA)15 microsatellite probe. Our results showed that B chromosomes self-pair in cells containing only one B chromosome. In cells with two identical B chromosomes, these elements remain as separate synaptonemal complexes or close self-paired elements in the nucleus territory. Overall, we reveal that B chromosomes can escape meiotic silencing of unsynapsed chromatin through a self-pairing process, allowing expression of their own genes to facilitate regular meiosis resulting in fertile individuals. This behavior, also seen in other congeneric species, might be related to their maintenance throughout the evolutionary history of Psalidodon.

Cytogenetic and molecular characteristics of Potamotrygon motoro and Potamotrygon sp. (Chondrichthyes, Myliobatiformes, Potamotrygonidae) from the Amazon basin: Implications for the taxonomy of the genus.

The chromosomes of two freshwater stingrays, Potamotrygon motoro and Potamotrygon sp., from the Amazon River basin in Brazil were investigated using integrated molecular (cytochrome c oxidase subunit 1) and cytogenetic analyses. Potamotrygon motoro presented intraspecific variation in the diploid number, with 2n=66 in the females and 2n=65 in the males, while Potamotrygon sp. had a karyotype with 66 chromosomes, in both sexes. The C-banding revealed the presence of heterochromatic blocks accumulated in the centromeric region of all the chromosomes in both species. The FISH assays with 18S DNA probes highlighted the terminal region of three or four chromosome pairs in P. motoro and seven chromosomes in Potamotrygon sp. The rDNA 5S sequences were found in only one chromosomal pair in both species. The interspecific genetic distance based on the COI sequences, between P. motoro and Potamotrygon sp. from Amazon River was 10.8%, while that between the Amazonian P. motoro and Potamotrygon amandae from the Paraná River was 2.2%, and the genetic distance between Potamotrygon sp. and P. amandae was 11.8%. In addition to the new insights on the cytogenetics of the study species, the results of the present study confirmed the existence of heteromorphic sex-linked chromosomes in P. motoro.

Satellite DNA content of B chromosomes in the characid fish Characidium gomesi supports their origin from sex chromosomes.

The origin of supernumerary (B) chromosomes is clearly conditioned by their ancestry from the standard (A) chromosomes. Sequence similarity between A and B chromosomes is thus crucial to determine B chromosome origin. For this purpose, we compare here the DNA sequences from A and B chromosomes in the characid fish Characidium gomesi using two main approaches. First, we found 59 satellite DNA (satDNA) families constituting the satellitome of this species and performed FISH analysis for 18 of them. This showed the presence of six satDNAs on the B chromosome: one shared with sex chromosomes and autosomes, two shared with sex chromosomes, one shared with autosomes and two being B-specific. This indicated that B chromosomes most likely arose from the sex chromosomes. Our second approach consisted of the analysis of five repetitive DNA families: 18S and 5S ribosomal DNA (rDNA), the H3 histone gene, U2 snDNA and the most abundant satDNA (CgoSat01-184) on DNA obtained from microdissected B chromosomes and from B-lacking genomes. PCR and sequence analysis of these repetitive sequences was successful for three of them (5S rDNA, H3 histone gene and CgoSat01-184), and sequence comparison revealed that DNA sequences obtained from the B chromosomes displayed higher identity with C. gomesi genomic DNA than with those obtained from other Characidium species. Taken together, our results support the intraspecific origin of B chromosomes in C. gomesi and point to sex chromosomes as B chromosome ancestors, which raises interesting prospects for future joint research on the genetic content of sex and B chromosomes in this species.

Phylogenomics of the Neotropical fish family Serrasalmidae with a novel intrafamilial classification (Teleostei: Characiformes).

The Neotropical fish family Serrasalmidae comprises 16 extant genera and 101 species widespread through major Neotropical rivers with relevant importance for regional fisheries and aquaculture. The monophyly of Serrasalmidae and the recognition of three main clades are recurrent between morphological and molecular phylogenies. However, both intergeneric and interspecific relationships within each of those clades remain uncertain. Here, we used 81 terminals of 69 species (68%) and all 16 genera of Serrasalmidae to sequence 1553 loci of ultraconserved elements (UCEs), multiple nuclear loci widely applied in phylogenetic studies, and performed maximum likelihood, Bayesian, and species tree analyses. We obtained highly supported phylogenies in all applied methods corroborating the monophyly of Serrasalmidae and the three-clade hypotheses herein proposed as two subfamilies and two tribes: (Colossomatinae (Serrasalminae (Myleini + Serrasalmini))). Morphological features for each subfamily involve the absence (Colossomatinae) or presence (Serrasalminae) of a pre-dorsal spine. Morphological diagnoses among tribes include the pre-dorsal spine being continuous (Myleini) or discontinuous (Serrasalmini) relative to the first unbranched dorsal-fin ray. Our results highlight the complexity of the relationships especially the non-monophyly of Myleus, Mylesinus, Myloplus, Tometes, and Utiaritichthys within Myleini, as well as of Serrasalmus and Pristobrycon within Serrasalmini.

Molecular cytogenetic analyses reveal extensive chromosomal rearrangements and novel B chromosomes in Moenkhausia (Teleostei, Characidae).

The cytogenetic characteristics of five fish species of the Moenkhausia are described, based on the analysis of specimens collected in different headwater. All the species analyzed presented 2n=50 chromosomes. The C-banding revealed a similar distribution pattern of heterochromatic blocks in all the species, except Moenkhausia nigromarginata. The 5S rDNA sites were distributed on multiple chromosome pairs in all five species. Single and multiple histone H1 sites were observed in all the species, and histone H1 was shown to be co-located with the 18S rRNA gene in a single chromosome pair. The U2 snDNA gene was distributed at multiple sites in all the Moenkhausia species. The presence of B microchromosomes was confirmed in Moenkhausia forestii, while individuals of the three study populations of Moenkhausia oligolepis presented three morphologically distinct types of B chromosome. The chromosomal mapping of the 18S rDNA sites using the FISH technique revealed signals in the B chromosomes of M. forestii, while clusters of the H1 histone and U2 snDNA genes were found in the B chromosomes of M. forestii and M. oligolepis. The classical and molecular cytogenetic markers used in this study revealed ample variation in the Moenkhausia karyotypes, reflecting the dynamic nature of the chromosomal evolution.

Phylogenomic reappraisal of the Neotropical catfish family Loricariidae (Teleostei: Siluriformes) using ultraconserved elements.

Neotropical freshwaters host more than 6000 fish species, of which 983 are suckermouth armored catfishes of the family Loricariidae - the most-diverse catfish family and fifth most species-rich vertebrate family on Earth. Given their diversity and ubiquitous distribution across many habitat types, loricariids are an excellent system in which to investigate factors that create and maintain Neotropical fish diversity, yet robust phylogenies needed to support such ecological and evolutionary studies are lacking. We sought to buttress the systematic understanding of loricariid catfishes by generating a genome-scale data set (1041 loci, 328,330 bp) for 140 species spanning 75 genera and five of six previously proposed subfamilies. Both maximum likelihood and Bayesian analyses strongly supported the monophyly of Loricariidae. Our results also reinforced the established backbone of loricariid interrelationships: Delturinae as sister to all other analyzed loricariids, with subfamily Rhinelepinae diverging next, followed by Loricariinae sister to Hypostominae + Hypoptopomatinae. Previous DNA-based relationships within Hypostominae and Loricariinae were strongly supported. However, we evaluated for the first time DNA-based relationships among many Hypoptopomatinae genera and found significant differences with this subfamily's current genus-level classification, prompting several taxonomic changes. Finally, we placed our topological results within a fossil-calibrated temporal context indicating that early Loricariidae diversification occurred across the Cretaceous-Paleogene boundary ∼65 million years ago (Ma). Our study lays a strong foundation for future research to focus on relationships among species and the macroevolutionary processes affecting loricariid diversification rates and patterns.

Chromosomal polymorphism and molecular variability in the pearly razorfish Xyrichtys novacula (Labriformes, Labridae): taxonomic and biogeographic implications.

The pearly razorfish Xyrichtys novacula (Linnaeus, 1758) is a sedentary benthic species distributed in both sides of the Atlantic Ocean and in the Mediterranean Sea. Previous cytogenetic analysis reported different diploid numbers in samples from Italy, Venezuela and Brazil. This research aims to test the hypothesis that samples from American Atlantic coast and Mediterranean Sea belong to the same single evolutionary lineage, characterized by intra-specific chromosome polymorphism. To this purpose a cytogenetic and molecular (mitochondrial COI sequences) survey was undertaken. Results revealed the existence of three different pearly razorfish molecular lineages: one present in Mediterranean Sea and two in the central and south American area, which are characterized by different karyotypes. One of these lineages shows substantial intra-population chromosomal polymorphism (2n = 45-48) determined by Robertsonian fusions that produce large metacentric chromosomes. On the whole data suggest that specimens morphologically identified as X. novacula correspond to three cryptic species.

Development of microsatellite markers using next-generation sequencing for the fish Colossoma macropomum.

Tambaqui (Colossoma macropomum) is a fish species from the Amazon and Orinoco Rivers, with favorable characteristics to the cultivation system and great market acceptance in South America. However, the construction of a genetic map for the genetic improvement of this species is limited by the low number of molecular markers currently described. Thus, this study aimed to validate gene-associated and anonymous (non-genic) microsatellites obtained by next generation sequencing (RNA-seq and whole genome shotgun-WGS, respectively), for future construction of a genetic map and search for quantitative trait loci (QTL) in this species. In the RNA-seq data, the observed and expected heterozygosity (Ho and He) ranged from 0.09 to 0.73, and 0.09 to 0.85, respectively. In the WGS data, Ho and He ranged from 0.33 to 0.95, and 0.28 to 0.92, respectively. In general, the evaluation of 200 markers resulted in 45 polymorphic loci, of which 14 were gene-associated (RNA-Seq) and 31 were anonymous (WGS). Moreover, some markers were related to genes of the immune system, biological regulation/control and biogenesis. This study contributes to increase the number of molecular markers available for genetic studies in C. macropomum, which will allow the development of breeding programs assisted by molecular markers.

Origin of B chromosomes in the genus Astyanax (Characiformes, Characidae) and the limits of chromosome painting.

Eukaryote genomes are frequently burdened with the presence of supernumerary (B) chromosomes. Their origin is frequently investigated by chromosome painting, under the hypothesis that sharing the repetitive DNA sequences contained in the painting probes is a sign of common descent. However, the intragenomic mobility of many anonymous DNA sequences contained in these probes (e.g., transposable elements) adds high uncertainty to this conclusion. Here we test the validity of chromosome painting to investigate B chromosome origin by comparing its results for seven B chromosome types in two fish species genus Astyanax, with those obtained (1) by means of the physical mapping of 18S ribosomal DNA (rDNA), H1 histone genes, the As51 satellite DNA and the (AC)15 microsatellite, and (2) by comparing the nucleotide sequence of one of these families (ITS regions from ribosomal DNA) between genomic DNA from B-lacking individuals in both species and the microdissected DNA from two metacentric B chromosomes found in these same species. Intra- and inter-specific painting suggested that all B chromosomes that were assayed shared homologous DNA sequences among them, as well as with a variable number of A chromosomes in each species. This finding would be consistent with a common origin for all seven B chromosomes analyzed. By contrast, the physical mapping of repetitive DNA sequences failed to give support to this hypothesis, as no more than two B-types shared a given repetitive DNA. Finally, sequence analysis of the ITS regions suggested that at least some of the B chromosomes could have had a common origin.

Chromosomal Spreading of Microsatellites and (TTAGGG)n Sequences in the Characidium zebra and C. gomesi Genomes (Characiformes: Crenuchidae).

Sex chromosome evolution involves the accumulation of repeat sequences such as multigenic families, noncoding repetitive DNA (satellite, minisatellite, and microsatellite), and mobile elements such as transposons and retrotransposons. Most species of Characidium exhibit heteromorphic ZZ/ZW sex chromosomes; the W is characterized by an intense accumulation of repetitive DNA including dispersed satellite DNA sequences and transposable elements. The aim of this study was to analyze the distribution pattern of 18 different tandem repeats, including (GATA)n and (TTAGGG)n, in the genomes of C. zebra and C. gomesi, especially in the C. gomesi W chromosome. In the C. gomesi W chromosome, weak signals were seen for (CAA)10, (CAC)10, (CAT)10, (CGG)10, (GAC)10, and (CA)15 probes. (GA)15 and (TA)15 hybridized to the autosomes but not to the W chromosome. The (GATA)n probe hybridized to the short arms of the W chromosome as well as the (CG)15 probe. The (GATA)n repeat is known to be a protein-binding motif. GATA-binding proteins are necessary for the decondensation of heterochromatic regions that hold coding genes, especially in some heteromorphic sex chromosomes that may keep genes related to oocyte development. The (TAA)10 repeat is accumulated in the entire W chromosome, and this microsatellite accumulation is probably involved in the sex chromosome differentiation process and crossover suppression in C. gomesi. These additional data on the W chromosome DNA composition help to explain the evolution of sex chromosomes in Characidium.

Effects of re-stripping on the seminal characteristics of pacu (Piaractus mesopotamicus) during the breeding season.

Seminal characteristics in teleost fish with an annual reproductive period, such as pacu (Piaractus mesopotamicus), may vary during the breeding season. The sperm formed before the beginning of the spawning period may be stored for a long time, causing damage to the cells. Therefore, re-stripping may be an important way to eliminate the "old" and allow for the collection of "new" spermatozoids. In this study, we analyzed the seminal characteristics of hormonally induced pacu at the beginning, middle and end of the breeding season, and we analyzed samples from re-stripped males (stripped first at the beginning, re-stripped in the middle, and re-stripped again at the end of the season) during two breeding seasons. The sperm density, ionic composition, pH, and osmolality were similar among the groups. The semen volume, seminal plasma protein concentration and incidence of morphologically anomalous sperm increased over time. In addition, some parameters that are associated with good-quality semen decreased, such as sperm motility, viability and DNA integrity. Moreover, we observed a positive association among motility, viability and DNA integrity for sperm with elevated 11-ketotestosterone, but there was no such association for fshb or lhb mRNA levels in the pituitary. The semen that was obtained earlier (at the beginning) or from re-stripped males exhibited better characteristics than the other samples collected. In conclusion, collecting semen from pacu at the end of breeding season should be avoided; it is preferable to strip early and then re-strip later in the season, and this approach may be used for diverse aquaculture purposes.

First Description of supernumerary Chromosomes in Ictaluruspunctatus Rafinesque 1818 Reveals Active Ribosomal Genes in the B Complement.

The North American channel catfish Ictalurus punctatus Rafinesque 1818 is cultivated in the United States, Asia and Brazilian fish farms, and also utilized as a model species in aquaculture and genetic studies. In this work, cytogenetic analysis of . punctatus from Brazilian aquaculture revealed for the first time the presence of extra elements (supernumerary or B chromosomes) in this species. These elements were characterized as dot-like micro B chromosomes and were found in three individuals (varying from 0 to 1) and in one individual with higher incidence per cell (varying from 0 to 5; mean number of Bs per cell = 2.01). More specific cytogenetic techniques in this individual revealed 58 A chromosomes (standard complement) containing heterochromatic bands in the centromeric regions, a single Ag-NOR in a subtelocentric pair (also positive for 18S rDNA using the FISH technique) and multiple 5S rDNA clusters in three different subtelocentric chromosomes. Four B chromosomes were entirely Ag-NOR positive (also fully heterochromatic) and three presented 18S rDNA clusters by FISH. The occurrence of Ag-NOR and 18S ribosomal genes in both A and B chromosome complements may indicate an intraspecific origin for these extra chromosomes. Additionally, the terminal location of 18S ribosomal clusters in the Ag-NOR-bearing chromosomes and the presence of active NOR in the B chromosomes suggested that breakage events may be related to a possible recent origin of these extra elements. We suggest this data may be useful as cytogenetic information for future elucidation of the composition, origin and evolution of extra chromosomes in fishes.

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones.

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.