Karyotypes and chromosomal features of both minor and major ribosomal RNA genes (rDNA) were investigated in nine Iberian chondrostomine species by fluorescent in situ hybridization (FISH) with 5S and 45S rDNA probes. All species presented invariably diploid values of 2n = 50 and the characteristic leuciscin karyotype pattern with 6-7 metacentric (m), 15-16 submetacentric (sm) and 3-4 subtelo- to acrocentric (st/a) chromosome pairs. The largest chromosome pair of the set was st/a as typical of Leuciscinae and no heteromorphic chromosomes could be unequivocally associated to sex determination. Achondrostoma occidentale and Pseudochondrostoma willkommii were cytogenetically characterized for the first time while Achondrostoma arcasii and Iberochondrostoma lemmingii were revisited regarding previous karyotype descriptions. Remarkable variability in number and location was observed for both molecular chromosome markers, especially within Achondrostoma and Iberochondrostoma genera. Clusters of 5S rDNA were mostly terminally associated to st/a chromosomes varying from four to eight positive signals, whilst NOR sites directly detected by the 45S rDNA probe were identified in sm chromosomes varying from three to six independent clusters. Frequent population bottlenecks in Mediterranean-type semiarid habitats were hypothesized to explain not only such extensive polymorphism which seems unique among leuciscin cyprinids but also the increased probability of fixation of rDNA translocation events.

Understanding the mechanisms underlying diversification and speciation by introgressive hybridization is currently one of the major challenges in evolutionary biology. Here, the analysis of hybridization between two pairs of Iberian Leuciscinae provided new data on independent hybrid zones involving Achondrostoma oligolepis (AOL) and Pseudochondrostoma duriense (PDU), and confirmed the occurrence of hybrids between AOL and Pseudochondrostoma polylepis (PPO). A multilevel survey combining morphological, genetic and cytogenomic markers on a vast population screening successfully sorted the selected fishes as admixed. Results were similar in both AOL Â PDU and AOL Â PPO systems. Overall, hybrid morphotypes, cytogenomic data and genetic profiling indicated preferential backcrossing and suggested AOL as a major genomic contributor. Moreover, results implied AOL as more permissive to introgression than PDU or PPO. Although PDU-and PPO-like individuals appeared more resilient to genome modifications, AOL appeared to be more involved and affected by the ongoing hybridization events, as chromosomal translocations were only found in AOL-like individuals. All hybrids analysed evidenced extensive ribosomal DNA (rDNA) polymorphism that was not found in parental species, but usually seen falling within the range of possible parental combinations. Yet, transgressive phenotypes that cannot be explained by normal recombination, including more rDNA clusters than expected or the occurrence of syntenic rDNAs, were also detected. Present results proved rapid genomic evolution providing the genetic novelty for species to persist. In addition, although the ultimate consequences of such apparently extensive and recurrent events remain unknown, modern genome-wide methodologies are of great promise towards answering questions concerning the causes, dynamics and impacts of hybridization.

Leuciscine cyprinids possess a nearly invariant diploid number (2n = 50) with an extremely uniform karyotype comprising of 8 pairs of metacentric, 13-15 pairs of submetacentric and 2-4 pairs of subtelocentric (st) to acrocentric (a) chromosomes. The largest pair is characteristically an st/a element-the 'leuciscine' cytotaxonomic marker. Previously, the interspecific homology of this chromosome pair could not be assessed owing to the inability to produce euchromatic or serial banding patterns. In the present study, we used laser-microdissection (15-20 copies of the marker chromosome) to construct a whole chromosome probe (WCP) from the marker chromosome of the roach Rutilus rutilus to ascertain the interspecific homology of marker chromosomes by cross-species in-situ hybridization. WCP was hybridized to chromosomes of widely distributed (Abramis brama, Alburnoides bipunctatus, Alburnus alburnus, Aspius aspius, Ballerus ballerus, B. sapa, Blicca bjoerkna, Chondrostoma nasus, Leucaspius delineatus, Leuciscus leuciscus, L. idus, R. rutilus, Scardinius erythrophthalmus, Squalius cephalus, and Vimba vimba) and Iberian endemic species (Achondrostoma oligolepis, Iberochondrostoma almacai, I. lusitanicum, Pseudochondrostoma duriense, S. alburnoides and S. pyrenaicus). Cross-species in-situ hybridization to chromosomes of Phoxinus phoxinus, a representative of leuciscine sister lineage, showed the same pattern as in all of the leuciscins. The probe consistently hybridized to the distal part of the short arm of the marker chromosome, indicating sequence homology.

Mercury (Hg) is one of the major aquatic contaminants even though emissions have been reduced over the years. Despite the relative abundance of investigations carried out on Hg toxicity, there is a scarcity of studies on its DNA damaging effects in fish under realistic exposure conditions. This study assessed the Hg genotoxicity in Golden grey mullets (Liza aurata) at Laranjo basin, a particularly contaminated area of Ria de Aveiro (Portugal) well known for its Hg contamination gradient. (1) Fish were seasonally caught at Laranjo basin and at a reference site (S. Jacinto), and (2) animals from the reference site were transplanted and caged (at bottom and surface), for 3 days, in two different locations within Laranjo basin. Using the comet assay, blood was analyzed for genetic damage and apoptotic cell frequency. The seasonal survey showed greater DNA damage in the Hg-contaminated area for all sampling seasons excluding winter. The temporal variation pattern of DNA lesions was: summer approximately autumn > winter > spring. Fish caged at Laranjo also exhibited greater DNA damage than those caged at the reference site, highlighting the importance of gill uptake on the toxicity of this metal. No increased susceptibility to apoptosis was detected in either wild or caged fish, indicating that mercury damages DNA of blood cells by a nonapoptotic mechanism. Both L. aurata and the comet assay proved to be sensitive and suitable for genotoxicity biomonitoring in mercury-contaminated coastal systems.

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Pseudochondrostoma duriense and Pseudochondrostoma polylepis hybridize extensively with Achondrostoma oligolepis in natural populations. In this first survey, karyotypes were comparatively analyzed by C-, AgNOR- and CMA(3)-banding procedures in pure (non-introgressed) fish specimens. Leuciscinae pattern was evidenced in the three species: metacentrics and submetacentrics dominance, a big subtelo/acrocentric (marker) chromosome pair and a 2n = 50; small macro-structural differences were observed. Heterochromatin was centromere-associated. Exceptions were found at sm1 and st/a1 long arms and at m1, sm3 and sm6 short arms. The st/a1 band was telomeric in the straight-mouth nases and sub-terminal in A. oligolepis. Multiple NORs of heterochromatic nature were found in sm pairs of the three species. Signals were telomeric except for one pair in A. oligolepis. Two to four structural and two functional NORs were found in P. duriense and P. polylepis, and four to six structural and four functional NORs in A. oligolepis. Species-specific markers will prove useful in hybrid zones' cytogenetic characterization and for in-depth studies of genome compatibility-related issues in future studies.

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Iberian Leuciscinae have been used in many studies as models to explore topics such as hybridization, allopolyploidy, modes of reproduction, and evolution. This article focuses on the contribution of cytogenomics to foster research in this group of cyprinid fish. Conventional and molecular banding results were reviewed, facilitating comparative analysis between nase and chub taxa inhabiting Portuguese freshwaters. Hybridization is known to occur within both Chondrostoma s.l. and Squalius genera although polyploidy has only been reported in the latter; the reasons behind such differential genome flexibility remain unidentified. FISH tools allowed recognizing additional chromosome markers, confirming NOR polymorphism and distinguishing species and their hybrids. Recombination and genome instability were detected in homoploid and polyploid hybrid genomes supporting active NOR transposition. However, the multiplication of rDNAs in these species does not seem to be associated with Rex3 retroelement, though hybrids were not surveyed. CGH and GISH allowed reaffirming the hybrid origin of S. alburnoides and confirming that the conservative karyotype patterns within Iberian leuciscines are restricted to the macrostructure. Current data also support the usefulness of mapping repetitive DNAs, especially for nonmodel compact genomes with less variable karyotypes and sequence data resources unavailable, like in many cyprinid lineages.