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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.

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.

Chromosomal locations of major ribosomal sites, i.e. NOR-phenotypes, were assigned in Squalius alburnoides complex using sequential chromomycin A3 (CMA3)- and silver (Ag)-staining. This hybridogenetic Iberian minnow comprises diploid, triploid and tetraploid forms that arose by interspecific hybridisation between S. pyrenaicus and an unknown species. Inheritance of NOR patterns was studied by means of crossing experiments involving most diploid-polyploid forms of the S. alburnoides complex with identified specific genotype constitution. In all the specimens studied, the NORs were localised in the short arms of submetacentric chromosomes. Although S. pyrenaicus presented only one pair of NOR-bearing chromosomes, the data from experimental crosses evidenced that S. alburnoides complex was characterised by a multiple NOR phenotype composed of one chromosome pair with stable NORs and two chromosome pairs with NOR site polymorphism of presence/absence type. These data suggest that the karyotype of the unknown parental species of the S. alburnoides complex should have a multiple NOR pattern and emphasised the role of the all-male diploid linage in the dynamics and evolutionary potential of the S. alburnoides complex allowing the preservation of the missing ancestor genome. Cross-analyses evidenced that in spite of the high polymorphic nature of NORs in this fish complex, we have no reason to reject the hypothesis that their inheritance patterns were in accordance with Mendelian segregation.

Two species of Odontocheila, O. confusa and O. nodicornis, from the Neotropical Region were studied regarding their karyotypes, localisation and activity of ribosomal genes and C-banding. The species, although belonging to the same genus, have quite distinct karyotypes. O. confusa has 10 pairs of autosomes and a single sex chromosome mechanism of the XY/XX type, thus a diploid value of 2n = 22 in males and females. One aneuploid male with a diploid number of 2n = 20 and one male with three B chromosomes were found in a total of eight males studied. O. nodicornis has 17 autosomal pairs and also a single chromosome system but of the X0/XX type, thus a diploid value of 2n = 35 in males and 2n = 36 in females. Fluorescence in situ hybridisation (FISH) revealed the presence of rDNA clusters in two autosomes in both species in mitotic and meiotic figures. Silver staining of male interphase nuclei confirmed the FISH results and showed that all rDNA genes were active. C-banding analysis revealed the presence of constitutive heterochromatin in the centromeres of all chromosomes in the two species plus two pairs in O. nodicornis with terminal positive C-bands. These results are discussed from the cytogenetic and evolutionary point of view.

The Iberian minnow Leuciscus alburnoides represents a complex of diploid and polyploid forms with altered modes of reproduction. In the present paper, we review the recent data on the origin, reproductive modes, and inter-relationships of the various forms of the complex, in order to predict its evolutionary potential. The complex follows the hybrid-origin model suggested for most other asexual vertebrates. Diploid and triploid females from the southern river basins exhibit reproductive modes that cannot be conveniently placed into the categories generally recognised for these vertebrate complexes, which imply continuous shifting between forms, where genomes derived from both parental ancestors are cyclically lost, gained or replaced. Replacement of nuclear genomes allow the introduction of novel genetic material, that may compensate for the disadvantages of asexual reproduction. Contrasting with most other vertebrate complexes, L. alburnoides males are fertile and play an important role in the dynamics of the complex. Moreover, diploid hybrid males may have initiated a tetraploidization process, when a diploid clonal sperm fertilised a diploid egg. This direct route to tetraploidy by originating fish with the right constitution for normal meiosis (symmetric), may eventually lead to a new sexually reproducing polyploid species. This case-study reinforces the significance of hybridisation and polyploidy in evolution and diversification of vertebrates.

Chromosomal polymorphism regarding the number of chromosomal NOR sites in the cyprinid fish Chondrostoma lusitanicum reported previously (Rodrigues & Collares-Pereira, 1996) was re-examined using fluorescence in situ hybridization (FISH) with a ribosomal DNA (rDNA) probe. All positive CMA3-bands contained ribosomal DNA documented by either two or four FISH positive signals in the respective karyotypes. This polymorphism suggests the occurrence of structural rearrangements of translocation type in rDNA region from one ancestral NOR-bearing chromosome pair ubiquitous among leuciscine cyprinid fishes to another pair. The absence of individuals heterozygous for this polymorphism is discussed.

Cellular suspensions of germinal tissues of Cylindera trisignata provided the definition of its karyotype: 2n = 23 and 2n = 26 for the males and females respectively. This Palearctic species has a sex chromosome system of the X1X2X3X4Y/X1X1X2X2X3X3X4X4 type, only found until now in Cicindela maroccana pseudomaroccana. The heterosomes are not well-differentiated from the general morphological standpoint. To explain the origin of the 4X condition, a mechanism of dissociation of the X chromosomes rather than an incorporation of autosomal segments is proposed. However, based on the occurrence of distinct ploidy levels, both in male and female cells, with a relatively high incidence for the tetraploid condition with regular autosomal bivalents, the hypothesis of a possible evolutionary role of polyploidy is suggested.