Introgressive hybridization as a promoter of genome reshuffling in natural homoploid fish hybrids (Cyprinidae, Leuciscinae).

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 × PDU and AOL × 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.

Chromosomes of Iberian Leuciscinae (Cyprinidae) revisited: Evidence of genome restructuring in homoploid hybrids using dual-color FISH and CGH.

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.

Molecular cytogenetics in artificial hybrid and highly polyploid sturgeons: An evolutionary story narrated by repetitive sequences.

We applied comparative genomic hybridization (CGH) and genomic in situ hybridization (GISH) to examine genomes of artificially produced sturgeon hybrids between sterlet, Acipenser ruthenus female (∼120 chromosomes) or Russian sturgeon, A. gueldenstaedtii female (∼240 chromosomes) and a spontaneous triploid Siberian sturgeon A. baerii male (∼360 chromosomes), respectively. The ploidy levels of progenies were analyzed by karyotyping and flow cytometry. We found that the species-specific regions were surprisingly identifiable only on some micro- and small(er) macrochromosomes in hybrid metaphases. We hypothesize that these distinguishable regions are represented by species-specific repetitive sequences driven by more dynamic molecular evolutionary mechanisms. On larger chromosomes, GISH faintly visualized only blocks of pericentromeric and telomeric repetitive sequences, remaining regions were equally shared by both parental species. We concluded that the interspecies hybridization producing viable and even fertile progeny is enabled by the fact that genomes of the species involved are likely divergent at the level of the repetitive sequences only and probably highly conserved in the coding sequences. These small differences of coding sequences are in concordance with previous estimations of relatedness of examined species producing artificial as well as natural hybrids. CGH and GISH represent a challenge in sturgeon cytogenetics as a valuable though technically not simple tool to discriminate chromosomes of parental species in hybrids. The potentials and drawbacks of CGH and GISH application in sturgeons are discussed and further experimental possibilities are proposed.

Chromosome studies of European cyprinid fishes: cross-species painting reveals natural allotetraploid origin of a Carassius female with 206 chromosomes.

A single female with 206 chromosomes and another 26 females with 156 chromosomes identified as Prussian carp, Carassius gibelio, and 5 individuals with 100 chromosomes identified as crucian carp, C. carassius, were sampled during field survey in one locality in the upper Elbe River. To identify the origin of females with high chromosome numbers, comparative karyotype analysis, GISH, with whole C. carassius DNA as probe and phylogenetic positions of sampled individuals revealed by cytochrome b mitochondrial marker were performed. GISH showed consistently bright labeling of 50 chromosomal elements out of 206, corresponding to the haploid chromosome number of C. carassius. The position of these females with high chromosome numbers in a reconstructed phylogenetic tree was within the clade of C. gibelio, documenting its affiliation to C. gibelio mitochondrial, i.e. maternal lineage. Our findings indicated that the mother of the female with high chromosome numbers was a gynogenetically reproducing 156-chromosome C. gibelio female and the father a bisexually reproducing C. carassius male. We, therefore, hypothesized that the C. gibelio × C. carassius allopolyploid female with 206 chromosomes arose by a mechanism of sperm genome addition to an unreduced egg of the mother.

Evidence for integrity of parental genomes in the diploid hybridogenetic water frog Pelophylax esculentus by genomic in situ hybridization.

The Western Palearctic water frogs Pelophylax ridibundus and P. lessonae were identified as parental (sexual) species and P. esculentus as their interspecific, hybridogenetically reproducing hybrid with hemiclonal heredity. We used genomic in situ hybridization (GISH) to identify parental chromosomes of P.lessonae and P.ridibundus in diploid P. esculentus karyotypes (2n = 26). GISH probes were made by fluorochrome labeling of total genomic DNA extracted from the sexual progenitors. The labeled probe from one species was hybridized to chromosomes of P. esculentus in the presence of excess of unlabeled genomic DNA from the other species. Thus, the P. lessonae probe was blocked by P. ridibundus unlabeled DNA, and vice versa. We successfully discriminated each of the 13 respective parental chromosomes in metaphase complements of the hybrids according to species-specific hybridization signals. GISH enabled us to confirm additional differences between parental chromosomes in size (smaller chromosomes belong to P. lessonae) and in the presence of DAPI-positive centromeric heterochromatin (detected in chromosomes of P. ridibundus, but not in P. lessonae). The fact that no visible intergenomic exchanges were found in metaphase chromosomes of diploid P. esculentus provides important information on the genomic integrity of hemiclonal transmission and supports hybridogenesis as a reproductive mode at the chromosome level for the specimens examined.

Sympatric occurrence of triploid, aneuploid and tetraploid weatherfish Misgurnus fossilis (Cypriniformes, Cobitidae).

Ploidy analyses of 116 weatherfish Misgurnus fossilis individuals revealed the sympatric occurrence of triploid, intermediate aneuploid and tetraploid specimens in a 1:1:4 ratio. No diploids were detected and the sex ratio of triploids and tetraploids was 1:1, while that of aneuploids was skewed at 3:1 for males. An origin of intermediate aneuploids from mating triploids with tetraploids is hypothesized.

Evidence for two transferrin loci in the Salmo trutta genome.

To determine the organization of transferrin (TF) locus in the Salmo trutta genome, partial DNA and cDNA sequencing, fluorescent in situ hybridization (FISH) and Salmo salar BAC analysis were performed. TF expression levels and copy number prediction were assessed using real-time PCR. In addition to two previously reported DNA TF variant sequences of S. trutta and Salmo marmoratus (TF1), two novel variant sequences (TF2) were revealed in both species. Variant-specific sequence tags, characterizing two variants for each TF type (TF1 and TF2), were identified in genomic clones from each of the F1 hybrids between S. trutta and S. marmoratus. These clearly documented double heterozygote status at the TF loci. The real-time PCR data showed that each of the two TF types (TF1 and TF2) existed in one copy only and that the transcription of TF2 was considerably lower compared with TF1. Using FISH, hybridization signals were observed on two medium-sized acrocentric chromosomes of S. trutta karyotype. A TF type-specific PCR followed by a restriction analysis revealed the presence of two TF loci in the majority of analysed BAC clones. It was concluded that the TF gene is duplicated in the genome of S. trutta, and that the two TF loci are located adjacent to one another on the same chromosome. The differing transcription levels of TF1 and TF2 appear to depend on the corresponding promoter activity, which at least for TF2 seems to vary between different Salmo congeners.

Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). III: extensive karyotypic variability associated with low mitochondrial haplotype differentiation in C. bivittatum.

We investigated chromosomal evolution in the African killifish species Chromaphyosemion bivittatum using a combination of cytogenetic and phylogenetic methods. Specimens from five populations were examined by conventional Giemsa staining as well as sequential chromosome banding with 4',6-diamidino-2-phenylindole (DAPI), chromomycin A(3) (CMA(3)), AgNO(3)-staining and C-banding. The cytogenetic analysis revealed variability in 2n ranging from 2n = 29 to 2n = 36 and in NF ranging from NF = 38 to NF = 44. Two populations showed an extensive chromosomal polymorphism (2n = 29-34, NF = 44 and 2n = 32-34, NF = 38-42, respectively). Karyotypic variability within and among populations was mainly due to Robertsonian translocations and heterochromatin additions, and chromosome banding patterns suggested that both types of chromosomal rearrangements were related to the presence of AT-rich heterochromatin. A phylogenetic analysis of the partial mitochondrial (mt) cytochrome b gene, using specimens from eleven populations, revealed a low degree of haplotype differentiation, which suggested a relatively recent divergence of the populations examined. This finding conformed to the low degree of morphological differentiation observed among C. bivittatum populations and might indicate fast chromosomal evolution. The high karyotypic variability may be caused by an elevated chromosomal mutation rate as well as certain aspects of the mating system and population dynamics of C. bivittatum facilitating the fixation of new chromosomal variants.

Karyotype and chromosomal characteristics of Ag-NOR sites and 5S rDNA in European smelt, Osmerus eperlanus.

Karyotype and cytogenetic characteristics of European smelt Osmerus eperlanus were investigated using different staining techniques (sequential Ag-, CMA3 and DAPI banding) and PRINS to detect 5S rDNA and telomeric sites. The diploid chromosome number was invariably 2n = 56 and karyotype composed of 5 pairs of metacentrics, 9 pairs of subtelocentrics and 14 pairs of subtelo- to acrocentrics. The DAPI-positive heterochromatic regions were found in centromeric positions on bi-armed chromosomes and few acrocentrics. Additionally, some interstitial DAPI-positive bands were identified on three pairs of submetacentric chromosomes. The nucleolar organizer regions (NORs) were detected in the short (p) arms of the largest metacentric pair of chromosomes No. 1. Sequential banding (Giemsa-, AgNO(3) and CMA(3) stainings) revealed NOR sites corresponding to achromatic regions but not associated with CMA(3)-positive blocks of heterochromatin located on either side of NORs. Individuals from the analyzed population had this conspicuous pair of chromosomes always in heterozygous combination. A complex inversion system was hypothesized to be involved in the origin of the observed variation but analysis with telomeric PRINS and PNA-FISH did not reveal any Interstitial Telomeric Sites (ITS). Hybridization signals were confined exclusively to terminal chromosomal regions. The 5S ribosomal sites as revealed by PRINS were found to be invariably located in the short (p) arms of four pairs of subtelocentric chromosomes. Cytotaxonomic comparisons of the present results with the voluminous available cytogenetic data-set from salmoniform and esociformes fishes appear to support the recent view, based on robust molecular-based phylogeny, that salmoniform and osmeriform fishes are not as closely related as previously assumed.

Karyotype differentiation in Chromaphyosemion killifishes (Cyprinodontiformes, Nothobranchiidae). II: cytogenetic and mitochondrial DNA analyses demonstrate karyotype differentiation and its evolutionary direction in C. riggenbachi.

African killifishes of the genus Chromaphyosemion show a high degree of phenotypic and karyotypic diversity. The latter is especially pronounced in C. riggenbachi, a morphologically defined species restricted to a small distribution area in Cameroon. This study presents a detailed reconstruction of karyotype differentiation within C. riggenbachi using conventional Giemsa staining and sequential chromosome banding as well as a phylogenetic analysis based on part of the mitochondrial (mt) cytochrome b gene from eleven populations. The cytogenetic analysis revealed differences in chromosome morphology, banding patterns and/or diploid chromosome number (2n) among all populations examined. Diploid number ranged from 2n = 20 to 2n = 36 and varied mainly among populations, while C-banding patterns and NOR phenotypes showed fixed differences among populations as well as some variability within populations. The mtDNA analysis disclosed five clearly differentiated haplotype groups. Mapping the karyotype data onto the mtDNA dendrogram revealed a decrease in 2n from the most basal to the most derived groups, thus demonstrating a reduction of 2n during their evolutionary history. Our results indicate that karyotype differentiation involved Robertsonian fusions as well as non-Robertsonian processes. Causes of the high karyotypic variability may include an elevated chromosomal mutation rate as well as certain features of the ecology and mating system that could facilitate the fixation of chromosomal rearrangements. The pattern of karyotype and haplotype differentiation and the results of previous crossing experiments suggest incipient speciation in C. riggenbachi.

Ice age cloning--comparison of the Quaternary evolutionary histories of sexual and clonal forms of spiny loaches (Cobitis; Teleostei) using the analysis of mitochondrial DNA variation.

Recent advances in population history reconstruction offered a powerful tool for comparisons of the abilities of sexual and clonal forms to respond to Quaternary climatic oscillations, ultimately leading to inferences about the advantages and disadvantages of a given mode of reproduction. We reconstructed the Quaternary historical biogeography of the sexual parental species and clonal hybrid lineages within the Europe-wide hybrid complex of Cobitis spiny loaches. Cobitis elongatoides and Cobitis taenia recolonizing Europe from separated refuges met in central Europe and the Pontic region giving rise to hybrid lineages during the Holocene. Cobitis elongatoides due to its long-term reproductive contact with the remaining parental species of the complex--C. tanaitica and C. spec.--gave rise to two clonal hybrid lineages probably during the last interglacial or even earlier, which survived the Würmian glaciation with C. elongatoides. These lineages followed C. elongatoides postglacial expansion and probably decreased its dispersal rate. Our data indicate the frequent origins of asexuality irrespective of the parental populations involved and the comparable dispersal potential of diploid and triploid lineages.

Evolutionary history of asexual hybrid loaches (Cobitis: Teleostei) inferred from phylogenetic analysis of mitochondrial DNA variation.

Reconstruction of the evolutionary history of asexual lineages undermines their suitability as models for the studies of evolutionary consequences of sexual reproduction. Using molecular tools we addressed the origin, age and maternal ancestry of diploid and triploid asexual lineages arisen through the hybridization between spiny loaches Cobitis elongatoides, C. taenia and C. tanaitica. Reconstructions of the phylogenetic relationships among mitochondrial DNA (mtDNA) haplotypes, revealed by sequence analyses, suggest that both hybrid complexes (C. elongatoides-taenia and C. elongatoides-tanaitica) contained several asexual lineages of independent origin. Cobitis elongatoides was the exclusive maternal ancestor of all the C. elongatoides-tanaitica hybrids, whereas within the C. elongatoides-taenia complex, hybridization was reciprocal. In both complexes the low haplotype divergences were consistent with a recent origin of asexual lineages. Combined mtDNA and allozyme data suggest that the triploids arose through the incorporation of a haploid sperm genome into unreduced ova produced by diploid hybrids.

Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae).

Karyotypic and cytogenetic characteristics of Vimba vimba and V. elongata were investigated using differential staining techniques (sequential C-banding, Ag- and CMA3-staining) and fluorescent in situ hybridization (FISH) with 28S rDNA probe. The diploid chromosome number in both species was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo- to acrocentrics. The largest chromosome pair of the complements was characteristically subtelo- to acrocentric. The nucleolar organizer regions (NORs) in both species were detected in the telomeres of a single, middle-sized subtelocentric chromosome pair, a pattern common in a number of other Leuciscinae. FISH with rDNA probe produced consistently positive hybridization signals detected in the same regions indicated by Ag-staining and CMA3-fluorescence. The distribution of C-positive heterochromatin was identical in both species, including a conspicuous size polymorphism of heterochromatic blocks in the largest metacentric and subtelo- to acrocentric chromosomal pairs. No heteromorphic sex chromosomes were detected. A single analyzed individual of V. melanops possessed the same karyotype and NOR phenotype as V. vimba and V. elongata. The apparent karyotype homogeneity and chromosomal characteristics of ribosomal DNA in all three species of the genus Vimba is consistent to that found in most other representatives of the European leuciscine cyprinid fishes.

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.

Chromosomal characteristics of ribosomal DNA in two extant species of North American mudminnows Umbra pygmaea and U. limi (Euteleostei: Umbridae).

The locations and chromosomal characteristics of ribosomal DNA (rDNA) sites in the karyotypes of two extant North American species of mudminnows, Umbra pygmaea and U. limi (2n = 22, NF = 44), were analyzed sequentially by conventional Giemsa staining, Ag staining, CMA(3) fluorescence and fluorescence in situ hybridization (FISH). The nucleolar organizer regions (NORs) were located in the fourth chromosomal pair in both species (pericentromeric region in U. pygmaea and subtelomeric in U. LIMI). These sites were strongly CMA(3)-positive suggesting that the rDNA sites in these species are associated with GC-rich DNA. FISH with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA(3)-fluorescence. However, both species also had additional CMA(3)-positive/Ag-negative heterochromatic blocks at pericentrometric regions of several chromosomal pairs (three in U. pygmaea and five in U. limi). FISH revealed additional rDNA clusters in both species. It is hypothesized that a paracentric inversion of the chromosome arm carrying the NORs might be one of the rearrangements differentiating the karyotypes of two North American species. The presence of additional rDNA sites is indicative of more complex rearrangements. The pericentromeric NOR phenotype of Umbra pygmaea is similar to that seen in U. krameri and in the distantly related genus Esox.

Chromosome evolution in the Salmonidae (Pisces): an update.

The karyotypes of salmonid fishes including taxa in the three subfamilies Coregoninae, Thymallinae and Salmoninae are described. This review is an update of the (Hartley, 1987) review of the chromosomes of salmonid fishes. As described in the previous review, the karyotypes of salmonid fishes fall into two main categories based on chromosome numbers: the type A karyotypes have diploid numbers close to 80 with approximately 100 chromosome arms (2n = 80, NF = 100), and the type B karyotypes have diploid numbers close to 60 with approximately 100 chromosome arms (2n = 60, NF = 100). In this paper we have proposed additional sub categories based on variation in the number of chromosome arms: the A' type with NF = 110-120, the A" type with NF greater than 140, and the B' type with NF less than 80. Two modes of chromosome evolution are found in the salmonids: in the Coregoninae and the Salmoninae the chromosomes have evolved by centric fusions of the Robertsonian type decreasing chromosome numbers (2n) while retaining chromosome arm numbers (NF) close to that found in the hypothetical tetraploid ancestor so that most extant taxa have either type A or type B karyotypes. In the Thymallinae, the chromosomes have evolved by inversions so that chromosome arm numbers (NF) have increased but chromosome numbers (2n) close to the karyotype of the hypothetical tetraploid ancestor have been retained and all taxa have type A' karyotypes. Most of the taxa with type B karyotypes in the Coregoninae and Salmoninae are members of the genus Oncorhynchus, although at least one example of type B karyotypes is found in all of the other genera. These taxa either have an anadromous life history or are found in specialized lacustrine environments. Selection for increases or decreases in genetic recombination as proposed by Qumsiyeh, 1994 could have been involved in the evolution of chromosome number in salmonid fishes.

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH).

When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA3-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n = 50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA3-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants - (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA3-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.

Ribosomal genes in Coregonid fishes (Coregonus lavaretus, C. albula and C. peled) (Salmonidae): single and multiple nucleolus organizer regions.

Major rDNA loci, i.e. nucleolus-organizing regions (NORs), were assigned using chromomycin-A3 (CMA3) staining followed by sequential silver (Ag) staining and in situ hybridization (ISH) with a rDNA probe to the chromosomes of the European whitefish (Coregonus lavaretus), the peled (Coregonus peled) and the vendace (Coregonus albula), three closely related coregonine salmonid fishes. One pair of NOR-bearing chromosomes was found in the peled karyotype. Multichromosomal, but stable, locations of rDNA sites on three pairs of chromosomes were observed in the European whitefish karyotype. Multichromosomal polymorphic locations, both in site and number, were observed in the karyotype of the vendace. Several Ag-, CMA3- and ISH-positive regions were found which defined up to seven cytotypes of five NOR-bearing chromosomes. All positive Ag-NORs detected corresponded both to rDNA-ISH- and CMA3-positive signals, which suggests extensive structural polymorphism in the locations of rDNA sites. Stable NOR sites were found at the same location on both homologous elements of the chromosome no. 9 in all individuals, while the remaining NORs were quite variable between individuals, and often present in heterozygous condition. The apparently similar and parallel evolutionary rDNA differentiation patterns in the subfamilies Coregoninae and Salmoninae (family Salmonidae) are observed and discussed.

Chromosomal characteristics of ribosomal DNA in the primitive semionotiform fish, longnose gar Lepisosteus osseus.

The chromosomes of longnose gar, Lepisosteus osseus, an extant representative of early radiation of actinopterygian fishes, were studied using conventional Giemsa-staining, Ag-staining, CMA3-fluorescence and fluorescence in-situ hybridization (FISH). The diploid chromosome number was 2n = 56 and the karyotype contained 11 pairs of metacentric, 6 pairs of submetacentric, 3 pairs of subtelocentric macrochromosomes and 16 microchromosomes. Nearly all macrochromosomes showed large CMA3-positive regions resembling the R-bands of higher vertebrates, indicating extensive distribution of GC-rich DNA along chromosomes. The nucleolar organizer regions (NORs) were located on the end of the short arm of a single small metacentric macrochromosomal pair. These sites were strongly CMA3-positive, suggesting that ribosomal sites are associated with GC-rich DNA. In-situ hybridization (FISH) with a rDNA probe gave consistently positive signals in the same regions detected by Ag-staining and CMA3-fluorescence. The evolutionary conservation of positive CMA3-fluorescence of ribosomal sites in 'holostean' and teleostean fishes is discussed.